JOURNAL
OF INVERTEBRATE
PATHOLOGY
s&272-277
(1990)
Response of Nuclear Polyhedrosis Virus-Resistant Spodoptera frugiperda Larvae to Other Pathogens and to Chemical Insecticides’ J. R. FUXA AND A. R. RICHTER Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 Received April 10, 1989; accepted June 22, 1989 Selection in the laboratory for Spodopterafrugiperda (Sf) resistant to nuclear polyhedrosis virus (NPV) affected the susceptibility of the insect to certain other mortality agents, including a chemical insecticide. Median lethal concentrations (L&s) and associated statistics were compared for several mortality agents between colonies of NPV-resistant and -susceptible (control) insects. Compared to the susceptible insects, the NPV-resistant insects were cross-resistant to the S. frugiperda granulosis virus and to the Autographa californica NPV based on nonoverlap of 95% fiducial limits of the LC,s. The NPV-resistant insects were significantly more susceptible to methyl parathion than the control insects. The two colonies of S. frugiperda did not differ significantly in their response to Bacillus thuringiensis, Vairimorpha necatrix, or carbaryl. The crossresistance experiments were based on per OSexposure of the insects to the pathogens and insecticides; the susceptibility of the resistant and control insects did not differ significantly when the Sf NPV was injected into the hemocoel or when methyl parathion was applied topically. o 1990 Academic
Press, Inc.
WORDS: Spodoptera frugiperda; nuclear polyhedrosis virus; granulosis virus; Spodoptera frugiperda NPV; Spodoptera frugiperda GV; Autographa californica NPV; Vairimorpha necatrix; Bacillus thuringiensis; methyl parathion; resistance; cross-resistance; resistance, negatively correlated. KEY
INTRODUCTION There have been few studies of crossresistance by insects between entomopathogens or between entomopathogens and chemical insecticides. Watanabe (1987) observed correlations in susceptibility of Bombyx mori to a nuclear polyhedrosis virus (NPV) and cytoplasmic polyhedrosis virus (CPV), but not between CPV and infectious flacherie virus. Similarly, one population of fall armyworm, Spodopteru frugiperda (Sf), was significantly less susceptible than another population to NPVs from three different insect species (Reichelderfer and Benton, 1974). Strains of Heliothis virescens that were susceptible or resistant to chemical insecticides did not significantly differ in their susceptibility to a bacterium, virus, fungus, or protozoan (Ig’ Approved for publication by the Director of the Louisiana Agricultural Experiment Station as Manuscript No. 89-17-3194. 272 0022-2011/90 $1.50 Copyrieht 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
noffo and Roush, 1986). Plodia interpunctella that were “presumed” to be resistant to malathion appeared to be less susceptible than other populations to Bacillus thuringiensis (Kinsinger and McGaughey, 1979). The S. fiugiperdu NPV system is one of the best understood virus-insect systems with regard to resistance. Field populations of 5’. fiugiperdu differ significantly in their susceptibility to NPV (Reichelderfer and Benton, 1974; Fuxa, 1987; Fuxa et al., 1988). Sf resistant to NPV have been selected in the laboratory (Fuxa et al., 1988), and the resistance is known to be due to a single gene or genes that lack dominance (Reichelderfer and Benton, 1974). The resistance is unstable due to selection against the resistance gene(s) in the absence of NPV (Fuxa and Richter, 1989). Finally, cross-resistance has been observed in the only agents tested in this system; S. frugiperdu that were resistant to the Sf NPV also were resistant to the Autographa cali-
CROSS-RESISTANCE
NPV and to the Trichoplusia ni NPV (Reichelderfer and Benton, 1974). The purpose of our research was to confirm cross-resistance to A. californica NPV and to determine whether selection for NPV resistance in S. fiugiperda induced cross-resistance to S. frugiperda granulosis virus (GV) or to mortality agents other than viruses, including a bacterium, a protozoan, and representatives from two different classes of chemical insecticides.
fornica
MATERIALS
AND METHODS
Two colonies of S. frugiperda, one resistant and one susceptible to the Sf NPV, were exposed to several other mortality agents in a test for cross-resistance. The S. frugiperda originally were collected as larvae in a corn field near Hammond, Louisiana, in 1985. The insects were split into two colonies; one served as a control and the other was subjected to selection pressure from exposure to the Sf NPV, as reported previously (Fuxa et al., 1988). The selection pressure resulted in a colony of insects with a resistance ratio of >3 x , The mortality agents were obtained from a variety of sources. The Sf NPV and Sf GV were isolated from S. frugiperda collected near Hammond and produced by standard techniques in S. frugiperda larvae. Autographa californica NPV was obtained originally from A. M. Heimpel (U.S. Department of Agriculture), and Vairimorpha necatrix was obtained from W. M. Brooks (Department of Entomology, North Carolina State University); both were produced in larvae of T. ni. Viral polyhedra or capsules and V. necatrix spores were purified by homogenization of infected larvae, filtration through cheesecloth, and table-top centrifugation; they were then counted on a Petroff-Hausser bacteria counter under phase-contrast microscopy. Bacillus thuringiensis was obtained from the product Dipel (Abbott Laboratories, North Chicago, Illinois; 8.45 x lo6 III/ml). Two chemical insecticides were included in the experiments: the organophosphate methyl
IN
RESPONSE
TO NPV
273
parathion (99.9% technical) and the carbamate carbaryl(90%). In the initial cross-resistance experiments, the insects were exposed to the mortality agents by ingestion. The virus dosages were fed to the insects by a larval drinking technique (Hughes and Wood, 1981; Mitchell and Smith, 1985). Neonates less than 8 hr old were placed in sterile, plastic petri dishes and were encircled by droplets of water containing dye and appropriate concentrations of the viral polyhedra or capsules. Ingestion of the suspension was confirmed by observing the dye in the insect guts through a dissecting microscope. The insects were fed dosages of the bacterium, protozoan, and two chemicals by a diet contamination technique (Richter and Fuxa, 1984), because the neonates would not drink suspensions of these agents. Standard glass tubing (4.5 mm i.d.j was cut into lengths of 50 mm and filled one-third full of artificial diet (Greene et al., 1976, but without the microbial inhibitors methyl paraben, sorbic acid, and formaldehyde) by insertion of the tube into l-day-old diet, The diet end was sealed with wax. From the open end, the diet surface was inoculated with the appropriate dosage of the bacterium or protozoan in 1-4 ).~lof sterile distilled water with a syringe (Hamilton Model 701) on a repeating dispenser (Hamilton B600- 1j. The chemical insecticides were mixed into the diet during its preparation. One third-instar S. frugiperda was placed into the tube, which was then plugged with cotton. After the diet was consumed, the insects were transferred to individual 30-ml cups with diet and reared at 27°C at 80% RH, and with a 14-hr photoperiod. The insects treated by the neonatal drinking technique were similarly reared individually. All insects were observed periodically for mortality for 12 days. The initial experiments indicated that the NPV-resistant and -susceptible S. frugiperda differed in their susceptibility to certain of the agents, and therefore it became of interest to learn whether the in-
274
FUXA AND RICHTER
sects would continue to differ if they were exposed to the Sf NPV or to methyl parathion by a method other than ingestion. Methyl parathion was applied topically to fourth-instar S. fiugiperda in 1 p.1of 95% ethanol/insect with a 1Oql syringe (Hamilton). The Sf NPV was injected directly into the hemocoel of late fourth- or early fifthinstars (Stairs, 1980). Virions were freed from their polyhedral inclusion bodies by suspension in 0.007 M Na&O, (final concentration) for 1 hr. The suspension was then centrifuged at 10,000 rpm for 1 hr, and the pellet was resuspended in distilled water and filtered through a 0.45pm millipore filter. The viral suspension (5 ~1) was injected into the larvae with a Hamilton IO-p,1 syringe. Except for the NPV injections, mortality was evaluated by estimating log doseprobit (ldp) lines for each agent in each of the two colonies of S. frugiperdu. Data TABLE Loo DOSE-PROBIT Spodoptera
frugiperda
from two to five replicates were combined to estimate each ldp line, including five to seven doses (+control) and 20-30 larvae/ dose/replicate. There was never any mortality in the controls. Log dose-probit parameters were estimated with a probit analysis program for microcomputers (MicroProbit 3.0, T. C. Sparks and A. Sparks, Department of Entomology, Louisiana State University) based on the method of Finney (1971). Mortality caused by NPV injection was analyzed with a t test; there were four replicates with 40-86 insects/ treatment/replicate, and there was no mortality in control insects injected with sterile distilled water. RESULTS
Selection for resistance to the Sf NPV affected the susceptibility of the insects to certain other mortality agents, including a chemical insecticide (Table 1). The insects 1
PARAMETERS FOR VARIOUS BIOLOGICAL AND CHEMICAL MORTALITY AGENTS FED TO LARVAE FROM Two LABORATORY COLONIES, ONE SUSCEPTIBLE AND ONE SELECTED FOR RESISTANCE TO S. frugiperda NUCLEAR POLYHEDROSIS VIRUS
Susceptible insects Mortality agenta
LGob
95% Fiducial Limitsb
Slope (SE)
6.5
3.1-9.3
49.7
Resistant insects
LC,sb
95% Fiducial Limitsb
Slope (SE)
Resistance ratio’
1.9 (0.49)
19.2
16.0-22.5
2.0 (0.25)
3.0*
40.3-64.1
1.5 (0.17)
109.5
80.1-177.7
1.3 (0.19)
2.2*
13.0
8.1-19.1
1.4 (0.24)
29.2
21.3-40.2
1.3 (0.21)
2.2*
168.0
144.3-l%. 1
1.8 (0.11)
221.0
192.6254.6
2.0 (0.14)
1.3
S. frugiperda
NPV S. frugiperda
GV Autographa californica
NPV Vairimorpha necatrix Bacillus thuringiensis
Methyl parathion Carbaryl
27.5
23.632.7
1.8 (0.21)
27.1
23.7-31.7
1.8 (0.16)
1.0
5.22 16.78
4.85-5.59 15.1b18.72
2.79 (0.17) 2.34 (0.18)
0.75 18.95
0.73-0.77 17.63-20.59
10.11 (0.74) 4.53 (0.42)
0.1* 1.1
u NPV, nuclear polyhedrosis virus; GV, granulosis virus. b Units for viruses = NPV polyhedral inclusion bodies or GV capsules/insect, for V. necatrix = spores/mm* of diet, for B. thuringiensis = international units/mm2 of diet, and for the two chemicals = parts per million of diet. ’ RR = LC, in NPV-resistant insects/L& in susceptibles. * Significant difference between insects based on nonoverlap of 95% fiducial limits.
CROSS-RESISTANCE
IN RESPONSE
resistant to Sf NPV were significantly less susceptible than unselected insects to the Sf GV and to the A. culifornica NPV, based on nonoverlap of 95% fiducial limits. For all three viruses, the slope of the ldp line in the resistant insects was similar to that in the susceptible insects. The resistance ratios were relatively low, ranging from 2.2 to 3.0. The insects also differed significantly in their response to methyl parathion, but in this case the NPV-resistant insects were significantly more susceptible than the unselected insects to the insecticide (Table 1). The slopes of the ldp lines were greater in the insects that were more susceptible to the chemical, that is, the NPV-resistant insects (Table 1, Fig. 1); but the ldp lines from the two groups of insects did not overlap (Fig. 1). The NPV-resistant and susceptible insects did not differ significantly in their response to V. necatrix, B. thuringiensis, or carbaryl (Table 1). The insects responded differently when the Sf NPV was injected or methyl parathion was applied topically rather than in-
Log
Dose
(Methyl
TO NPV
275
gested. When the Sf NPV was injected into the hemocoel, there was 57.6% mortality (4 replicates, total n = 263, SE = 16.67) in the NPV-susceptible insects and 57.4% (4 replicates, total n = 203, SE = 16.26) in the NPV-resistant insects. These mortality percentages were not significantly different (t test, P < 0.05). When methyl parathion was applied topically, the LD,, was 0.14 &insect (95% fiducial limits = 0.13-0.15, slope = 2.81, SE,,,,p, = 0.21) in the NPVsusceptible insects and 0.13 )&insect (95% fiducial limits = 0.1 l-0.15, slope = 2.64, SE sl0pe = 0.29) in the NPV-resistant insects. Thus, these LD,,s were not significantly different based on overlap of 95% fiducial limits. DISCUSSION The ldp lines for the viruses and for methyl parathion in the two colonies of S. fiugiperdu suggest certain conclusions about susceptibility of the populations to the mortality agents. When insects are selected for resistance to a mortality agent in the labo-
Parathion.
PPM
of diet)
FIG. 1. Log dose-probit lines for methyl parathion fed in artiCal diet to Spodoprera frugiperda larvae from two laboratory colonies, one susceptible and one selected for resistance to nuclear polyhedrosis virus. Each line represents one replicate.
276
FUXA
AND
ratory, specific resistance is indicated when the ldp regression line initially flattens and then becomes steeper as the LD,, increases. If the LD,, increases with no change in slope, usually to a resistance ratio of ca. 2, then the insects are being selected for a more generalized response, vigor tolerance, as opposed to any specific resistance mechanism (Brown (and Pal, 1971). In the case of S. frugiperdu and its NPV, the initial and final lines were too close together to safely draw conclusions about slopes of intermediate lines (Table 1) (Fuxa et al., 1988), but there apparently was specific resistance since previous research had indicated that S. frugiperda carries a gene or genes for resistance that lack dominance (Reichelderfer and Benton, 1974). Specific resistance is further indicated by the fact that the insects were cross-resistant to other baculoviruses but not to B. thuringiensis or V. necatrix (Table 1) (Reichelderfer and Benton, 1974). The ldp lines for methyl parathion, on the other hand, are almost identical to classical ldp lines for the beginnings of specific resistance, except that the insects were being selected by the NPV in the reverse direction, that is, for increased rather than decreased susceptibility to the chemical (Fig. 1). Thus, as we were selecting insects for resistance to NPV, we were simultaneously eliminating the more methyl parathionresistant insects, thereby increasing the slope of the methyl parathion ldp line and reducing population variability with respect to methyl parathion susceptibility. The data resulting from injection of NPV and topical application of methyl parathion indicate that differences in susceptibility to both agents were due to factors associated with the gut of the insects. When the insects ingested the virus or chemical, there were differences in susceptibility (Table 1); but when the gut was bypassed by other methods of treatment, there were no differences (see under Results). Previous research has indicated that insect populations or individuals can vary in their ability to inactivate
RICHTER
viruses in the gut lumen, in their susceptibility to viral attachment or to replication in midgut epithelial cells, and in their ability to discharge and regenerate infected midgut cells (Briese, 1986). Research of insect gut as a site of action of organophosphate or carbamate insecticides has been limited mainly to penetration studies: there is variation among insect species in the penetration of insecticides through the gut, and parathion penetrates honeybee midgut more quickly than carbaryl (Brooks, 1976). Thus, it is possible that the differences in susceptibility to the NPV and to methyl parathion relate either to some effect of the gut contents on the agent or to penetration or invasion through the gut cells. The current results are likely to have practical significance only if the S. frugiperdu cross-resistance to biotic agents or negatively correlated resistance to chemicals, which have rarely been researched in other systems,are not isolated phenomena. Cross-resistance certainly can affect the use of chemical insecticides for insect control (Brown and Pal, 1971). However, most insect viruses are so host-specific that insectsgenerally are not subject to natural or manipulated epizootics by more than one virus. For example, in Louisiana the Sf NPV has been known to kill up to 68% of the larvae in a field population of S. frugiperdu; but the only other virus to which S. frugiperda are exposed is the Sf GV, which has not been observed to kill more than 7% (Fuxa, 1982). Populations of S.frugiperdu have developed resistance to methyl parathion (Pitre, 1986); thus, it seems feasible that extensive natural epizootics of nuclear polyhedrosis, such as occur in Louisiana (Fuxa, 1982), and differential NPV susceptibility in S. frugiperdu field populations (Fuxa, 1987; Fuxa et al., 1988) could improve control of the insect by methyl parathion. Many pestiferous insects that are subject to viral epizootics must often be controlled with chemical insecticides; and negatively correlated resistance, which occasionally is observed between chemical in-
CROSS-RESISTANCE
secticides, can provide opportunity for resistance management (Brown and Pal, 1971; Kurtak et al., 1987). However, the negatively correlated resistance in the current research was observed only in the feeding experiments and not by topical application, whereas methyl parathion acts largely as a contact insecticide in field applications . It initially is disturbing to those interested in microbial control to learn that resistance to insect viruses might be a widespread phenomenon (Briese, 1986; Fuxa et al., 1988). However, the reversion to susceptibility (Fuxa and Richter, 1989), the lack of cross-resistance to nonviral mortality agents, and the example of negatively correlated resistance to a chemical are positive features that perhaps can be exploited for resistance management in microbial control. It is clear that further research is necessary to determine whether these phenomena are widespread among different virus-insect systems. ACKNOWLEDGMENTS The authors thank Drs. Jerry B. Graves and Thomas C. Sparks for critically reading the manuscript and for providing helpful suggestions.
REFERENCES BRIESE, D. T. 1986. Insect resistance to baculoviruses. In “The Biology of Baculoviruses,” Vol. 2, “Practical Application for Insect Control” (R. R. Granados and B. A. Federici, Eds.), CRC Press, Boca Raton, FL. BROOKS, G. T. 1976. Penetration and distribution of insecticides. In “Insecticide Biochemistry and Physiology” (C. F. Wilkinson, Ed.), Plenum, New York/London. BROWN, A. W. A., AND PAL, R. 1971. “Insecticide Resistance in Arthropods.” World Health Organi~ation, Geneva. FINNEY, D. J. 1971. “Probit Analysis.” Cambridge Univ. Press, New York. FUXA, J. R. 1982. Prevalence of viral infections in populations of fall armyworm, Spodopreru frugiperda, in southeastern Louisiana. Environ. Entomol., 11, 239-242. FUXA, J. R. 1987. Spodoptera frugiperda susceptibil-
IN RESPONSE TO NPV
277
ity to nuclear polyhedrosis virus isolates with reference to insect migration. Environ. Entomol., 16, 218-223. FUXA, J. R., MITCHELL, F. L., AND RICHTER, A. R. 1988. Resistance of Spodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory. Entomophaga, 33, 55-63. FUXA, J. R., AND RICHTER, A. R. 1989. Reversion of resistance by Spodoptera frugiperda to nuclear polyhedrosis virus. J. Znvertebr. Pathol., 53, 52-56. GREENE, G. L., LEPPLA, N. C., AND DICKERSON, W. A. 1976. Velvetbean caterpillar: a rearing procedure and artificial medium. J. Econ. Entomol., 69, 487-488. HUGHES, P. R., AND WOOD, H. A. 1981. A synchronous peroral technique for the bioassay of insect viruses. J. Znvertebr. Puthol., 37, 154-159. IGNOFFO, C. M., AND ROLJSH, R. T. 1986. Susceptibility of permethrin- and methomyl-resistant strains of Heliothis virescens (Lepidoptera: Noctuidae) to representative species of entomopathogens, J. Econ. Entomol., 19, 334-337. KINSINGER, R. A., AND MCGAUGHEY, W. H. 1979. Susceptibility of populations of Indianmeal moth and almond moth to Bacillus thuringiensis. J. Econ. Entomol., 12, 346-349. KURTAK, D., MEYER, R., OCRAN, M., OUBDRAOGO, M., RENAUD, P., SAWADOGO, R. O., AND TBLI?, B. 1987. Management of insecticide resistance in control of the Simulium damnosum complex by the Onchocerciasis Control Programme, West Africa: Potential use of negative correlation between organophosphate resistance and pyrethroid susceptibility. Med. Vet. Entomol., 1, 137-146. MITCHELL, F. L., AND SMITH, J. W. 1985. Pathology and bioassays of the lesser comstafk borer (Elasmopalpus lignosellus) entomopoxvirus. J. Znvertebr. Pathol., 45, 75-80. PITRE, H. N. 1986. Chemical control of the fall armyworm (Lepidoptera: Noctuidae): An update. Florida Entomol., 69, 570-578. REICHELDERFER, C. F., AND BENTON, C. V. 1974. Some genetic aspects of the resistance of Spodoptera frugiperda to a nuclear polyhedrosis virus. J. Znvertebr. Pathol., 23, 378-382. RICHTER, A. R., AND FUXA, J. R. 1984. Pathogenpathogen and pathogen-insectide interactions in velvetbean caterpillar (Lepidoptera: Noctuidae). J. Econ. Entomol., 11, 1559-1564. STAIRS, G. R. 1980. Comparative infectivity of nonoceluded virions, polyhedra, and virions released from polyhedra for larvae of Galleria mellonella. J. Znvertebr. Pathol., 36, 281-282. WATANABE, H. 1987. The host population. In “Epizootiology of Insect Diseases” (J. R. Fuxa and Y. Tanada, Eds.). Wiley, New York.
OF INVERTEBRATE
PATHOLOGY
s&272-277
(1990)
Response of Nuclear Polyhedrosis Virus-Resistant Spodoptera frugiperda Larvae to Other Pathogens and to Chemical Insecticides’ J. R. FUXA AND A. R. RICHTER Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803 Received April 10, 1989; accepted June 22, 1989 Selection in the laboratory for Spodopterafrugiperda (Sf) resistant to nuclear polyhedrosis virus (NPV) affected the susceptibility of the insect to certain other mortality agents, including a chemical insecticide. Median lethal concentrations (L&s) and associated statistics were compared for several mortality agents between colonies of NPV-resistant and -susceptible (control) insects. Compared to the susceptible insects, the NPV-resistant insects were cross-resistant to the S. frugiperda granulosis virus and to the Autographa californica NPV based on nonoverlap of 95% fiducial limits of the LC,s. The NPV-resistant insects were significantly more susceptible to methyl parathion than the control insects. The two colonies of S. frugiperda did not differ significantly in their response to Bacillus thuringiensis, Vairimorpha necatrix, or carbaryl. The crossresistance experiments were based on per OSexposure of the insects to the pathogens and insecticides; the susceptibility of the resistant and control insects did not differ significantly when the Sf NPV was injected into the hemocoel or when methyl parathion was applied topically. o 1990 Academic
Press, Inc.
WORDS: Spodoptera frugiperda; nuclear polyhedrosis virus; granulosis virus; Spodoptera frugiperda NPV; Spodoptera frugiperda GV; Autographa californica NPV; Vairimorpha necatrix; Bacillus thuringiensis; methyl parathion; resistance; cross-resistance; resistance, negatively correlated. KEY
INTRODUCTION There have been few studies of crossresistance by insects between entomopathogens or between entomopathogens and chemical insecticides. Watanabe (1987) observed correlations in susceptibility of Bombyx mori to a nuclear polyhedrosis virus (NPV) and cytoplasmic polyhedrosis virus (CPV), but not between CPV and infectious flacherie virus. Similarly, one population of fall armyworm, Spodopteru frugiperda (Sf), was significantly less susceptible than another population to NPVs from three different insect species (Reichelderfer and Benton, 1974). Strains of Heliothis virescens that were susceptible or resistant to chemical insecticides did not significantly differ in their susceptibility to a bacterium, virus, fungus, or protozoan (Ig’ Approved for publication by the Director of the Louisiana Agricultural Experiment Station as Manuscript No. 89-17-3194. 272 0022-2011/90 $1.50 Copyrieht 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
noffo and Roush, 1986). Plodia interpunctella that were “presumed” to be resistant to malathion appeared to be less susceptible than other populations to Bacillus thuringiensis (Kinsinger and McGaughey, 1979). The S. fiugiperdu NPV system is one of the best understood virus-insect systems with regard to resistance. Field populations of 5’. fiugiperdu differ significantly in their susceptibility to NPV (Reichelderfer and Benton, 1974; Fuxa, 1987; Fuxa et al., 1988). Sf resistant to NPV have been selected in the laboratory (Fuxa et al., 1988), and the resistance is known to be due to a single gene or genes that lack dominance (Reichelderfer and Benton, 1974). The resistance is unstable due to selection against the resistance gene(s) in the absence of NPV (Fuxa and Richter, 1989). Finally, cross-resistance has been observed in the only agents tested in this system; S. frugiperdu that were resistant to the Sf NPV also were resistant to the Autographa cali-
CROSS-RESISTANCE
NPV and to the Trichoplusia ni NPV (Reichelderfer and Benton, 1974). The purpose of our research was to confirm cross-resistance to A. californica NPV and to determine whether selection for NPV resistance in S. fiugiperda induced cross-resistance to S. frugiperda granulosis virus (GV) or to mortality agents other than viruses, including a bacterium, a protozoan, and representatives from two different classes of chemical insecticides.
fornica
MATERIALS
AND METHODS
Two colonies of S. frugiperda, one resistant and one susceptible to the Sf NPV, were exposed to several other mortality agents in a test for cross-resistance. The S. frugiperda originally were collected as larvae in a corn field near Hammond, Louisiana, in 1985. The insects were split into two colonies; one served as a control and the other was subjected to selection pressure from exposure to the Sf NPV, as reported previously (Fuxa et al., 1988). The selection pressure resulted in a colony of insects with a resistance ratio of >3 x , The mortality agents were obtained from a variety of sources. The Sf NPV and Sf GV were isolated from S. frugiperda collected near Hammond and produced by standard techniques in S. frugiperda larvae. Autographa californica NPV was obtained originally from A. M. Heimpel (U.S. Department of Agriculture), and Vairimorpha necatrix was obtained from W. M. Brooks (Department of Entomology, North Carolina State University); both were produced in larvae of T. ni. Viral polyhedra or capsules and V. necatrix spores were purified by homogenization of infected larvae, filtration through cheesecloth, and table-top centrifugation; they were then counted on a Petroff-Hausser bacteria counter under phase-contrast microscopy. Bacillus thuringiensis was obtained from the product Dipel (Abbott Laboratories, North Chicago, Illinois; 8.45 x lo6 III/ml). Two chemical insecticides were included in the experiments: the organophosphate methyl
IN
RESPONSE
TO NPV
273
parathion (99.9% technical) and the carbamate carbaryl(90%). In the initial cross-resistance experiments, the insects were exposed to the mortality agents by ingestion. The virus dosages were fed to the insects by a larval drinking technique (Hughes and Wood, 1981; Mitchell and Smith, 1985). Neonates less than 8 hr old were placed in sterile, plastic petri dishes and were encircled by droplets of water containing dye and appropriate concentrations of the viral polyhedra or capsules. Ingestion of the suspension was confirmed by observing the dye in the insect guts through a dissecting microscope. The insects were fed dosages of the bacterium, protozoan, and two chemicals by a diet contamination technique (Richter and Fuxa, 1984), because the neonates would not drink suspensions of these agents. Standard glass tubing (4.5 mm i.d.j was cut into lengths of 50 mm and filled one-third full of artificial diet (Greene et al., 1976, but without the microbial inhibitors methyl paraben, sorbic acid, and formaldehyde) by insertion of the tube into l-day-old diet, The diet end was sealed with wax. From the open end, the diet surface was inoculated with the appropriate dosage of the bacterium or protozoan in 1-4 ).~lof sterile distilled water with a syringe (Hamilton Model 701) on a repeating dispenser (Hamilton B600- 1j. The chemical insecticides were mixed into the diet during its preparation. One third-instar S. frugiperda was placed into the tube, which was then plugged with cotton. After the diet was consumed, the insects were transferred to individual 30-ml cups with diet and reared at 27°C at 80% RH, and with a 14-hr photoperiod. The insects treated by the neonatal drinking technique were similarly reared individually. All insects were observed periodically for mortality for 12 days. The initial experiments indicated that the NPV-resistant and -susceptible S. frugiperda differed in their susceptibility to certain of the agents, and therefore it became of interest to learn whether the in-
274
FUXA AND RICHTER
sects would continue to differ if they were exposed to the Sf NPV or to methyl parathion by a method other than ingestion. Methyl parathion was applied topically to fourth-instar S. fiugiperda in 1 p.1of 95% ethanol/insect with a 1Oql syringe (Hamilton). The Sf NPV was injected directly into the hemocoel of late fourth- or early fifthinstars (Stairs, 1980). Virions were freed from their polyhedral inclusion bodies by suspension in 0.007 M Na&O, (final concentration) for 1 hr. The suspension was then centrifuged at 10,000 rpm for 1 hr, and the pellet was resuspended in distilled water and filtered through a 0.45pm millipore filter. The viral suspension (5 ~1) was injected into the larvae with a Hamilton IO-p,1 syringe. Except for the NPV injections, mortality was evaluated by estimating log doseprobit (ldp) lines for each agent in each of the two colonies of S. frugiperdu. Data TABLE Loo DOSE-PROBIT Spodoptera
frugiperda
from two to five replicates were combined to estimate each ldp line, including five to seven doses (+control) and 20-30 larvae/ dose/replicate. There was never any mortality in the controls. Log dose-probit parameters were estimated with a probit analysis program for microcomputers (MicroProbit 3.0, T. C. Sparks and A. Sparks, Department of Entomology, Louisiana State University) based on the method of Finney (1971). Mortality caused by NPV injection was analyzed with a t test; there were four replicates with 40-86 insects/ treatment/replicate, and there was no mortality in control insects injected with sterile distilled water. RESULTS
Selection for resistance to the Sf NPV affected the susceptibility of the insects to certain other mortality agents, including a chemical insecticide (Table 1). The insects 1
PARAMETERS FOR VARIOUS BIOLOGICAL AND CHEMICAL MORTALITY AGENTS FED TO LARVAE FROM Two LABORATORY COLONIES, ONE SUSCEPTIBLE AND ONE SELECTED FOR RESISTANCE TO S. frugiperda NUCLEAR POLYHEDROSIS VIRUS
Susceptible insects Mortality agenta
LGob
95% Fiducial Limitsb
Slope (SE)
6.5
3.1-9.3
49.7
Resistant insects
LC,sb
95% Fiducial Limitsb
Slope (SE)
Resistance ratio’
1.9 (0.49)
19.2
16.0-22.5
2.0 (0.25)
3.0*
40.3-64.1
1.5 (0.17)
109.5
80.1-177.7
1.3 (0.19)
2.2*
13.0
8.1-19.1
1.4 (0.24)
29.2
21.3-40.2
1.3 (0.21)
2.2*
168.0
144.3-l%. 1
1.8 (0.11)
221.0
192.6254.6
2.0 (0.14)
1.3
S. frugiperda
NPV S. frugiperda
GV Autographa californica
NPV Vairimorpha necatrix Bacillus thuringiensis
Methyl parathion Carbaryl
27.5
23.632.7
1.8 (0.21)
27.1
23.7-31.7
1.8 (0.16)
1.0
5.22 16.78
4.85-5.59 15.1b18.72
2.79 (0.17) 2.34 (0.18)
0.75 18.95
0.73-0.77 17.63-20.59
10.11 (0.74) 4.53 (0.42)
0.1* 1.1
u NPV, nuclear polyhedrosis virus; GV, granulosis virus. b Units for viruses = NPV polyhedral inclusion bodies or GV capsules/insect, for V. necatrix = spores/mm* of diet, for B. thuringiensis = international units/mm2 of diet, and for the two chemicals = parts per million of diet. ’ RR = LC, in NPV-resistant insects/L& in susceptibles. * Significant difference between insects based on nonoverlap of 95% fiducial limits.
CROSS-RESISTANCE
IN RESPONSE
resistant to Sf NPV were significantly less susceptible than unselected insects to the Sf GV and to the A. culifornica NPV, based on nonoverlap of 95% fiducial limits. For all three viruses, the slope of the ldp line in the resistant insects was similar to that in the susceptible insects. The resistance ratios were relatively low, ranging from 2.2 to 3.0. The insects also differed significantly in their response to methyl parathion, but in this case the NPV-resistant insects were significantly more susceptible than the unselected insects to the insecticide (Table 1). The slopes of the ldp lines were greater in the insects that were more susceptible to the chemical, that is, the NPV-resistant insects (Table 1, Fig. 1); but the ldp lines from the two groups of insects did not overlap (Fig. 1). The NPV-resistant and susceptible insects did not differ significantly in their response to V. necatrix, B. thuringiensis, or carbaryl (Table 1). The insects responded differently when the Sf NPV was injected or methyl parathion was applied topically rather than in-
Log
Dose
(Methyl
TO NPV
275
gested. When the Sf NPV was injected into the hemocoel, there was 57.6% mortality (4 replicates, total n = 263, SE = 16.67) in the NPV-susceptible insects and 57.4% (4 replicates, total n = 203, SE = 16.26) in the NPV-resistant insects. These mortality percentages were not significantly different (t test, P < 0.05). When methyl parathion was applied topically, the LD,, was 0.14 &insect (95% fiducial limits = 0.13-0.15, slope = 2.81, SE,,,,p, = 0.21) in the NPVsusceptible insects and 0.13 )&insect (95% fiducial limits = 0.1 l-0.15, slope = 2.64, SE sl0pe = 0.29) in the NPV-resistant insects. Thus, these LD,,s were not significantly different based on overlap of 95% fiducial limits. DISCUSSION The ldp lines for the viruses and for methyl parathion in the two colonies of S. fiugiperdu suggest certain conclusions about susceptibility of the populations to the mortality agents. When insects are selected for resistance to a mortality agent in the labo-
Parathion.
PPM
of diet)
FIG. 1. Log dose-probit lines for methyl parathion fed in artiCal diet to Spodoprera frugiperda larvae from two laboratory colonies, one susceptible and one selected for resistance to nuclear polyhedrosis virus. Each line represents one replicate.
276
FUXA
AND
ratory, specific resistance is indicated when the ldp regression line initially flattens and then becomes steeper as the LD,, increases. If the LD,, increases with no change in slope, usually to a resistance ratio of ca. 2, then the insects are being selected for a more generalized response, vigor tolerance, as opposed to any specific resistance mechanism (Brown (and Pal, 1971). In the case of S. frugiperdu and its NPV, the initial and final lines were too close together to safely draw conclusions about slopes of intermediate lines (Table 1) (Fuxa et al., 1988), but there apparently was specific resistance since previous research had indicated that S. frugiperda carries a gene or genes for resistance that lack dominance (Reichelderfer and Benton, 1974). Specific resistance is further indicated by the fact that the insects were cross-resistant to other baculoviruses but not to B. thuringiensis or V. necatrix (Table 1) (Reichelderfer and Benton, 1974). The ldp lines for methyl parathion, on the other hand, are almost identical to classical ldp lines for the beginnings of specific resistance, except that the insects were being selected by the NPV in the reverse direction, that is, for increased rather than decreased susceptibility to the chemical (Fig. 1). Thus, as we were selecting insects for resistance to NPV, we were simultaneously eliminating the more methyl parathionresistant insects, thereby increasing the slope of the methyl parathion ldp line and reducing population variability with respect to methyl parathion susceptibility. The data resulting from injection of NPV and topical application of methyl parathion indicate that differences in susceptibility to both agents were due to factors associated with the gut of the insects. When the insects ingested the virus or chemical, there were differences in susceptibility (Table 1); but when the gut was bypassed by other methods of treatment, there were no differences (see under Results). Previous research has indicated that insect populations or individuals can vary in their ability to inactivate
RICHTER
viruses in the gut lumen, in their susceptibility to viral attachment or to replication in midgut epithelial cells, and in their ability to discharge and regenerate infected midgut cells (Briese, 1986). Research of insect gut as a site of action of organophosphate or carbamate insecticides has been limited mainly to penetration studies: there is variation among insect species in the penetration of insecticides through the gut, and parathion penetrates honeybee midgut more quickly than carbaryl (Brooks, 1976). Thus, it is possible that the differences in susceptibility to the NPV and to methyl parathion relate either to some effect of the gut contents on the agent or to penetration or invasion through the gut cells. The current results are likely to have practical significance only if the S. frugiperdu cross-resistance to biotic agents or negatively correlated resistance to chemicals, which have rarely been researched in other systems,are not isolated phenomena. Cross-resistance certainly can affect the use of chemical insecticides for insect control (Brown and Pal, 1971). However, most insect viruses are so host-specific that insectsgenerally are not subject to natural or manipulated epizootics by more than one virus. For example, in Louisiana the Sf NPV has been known to kill up to 68% of the larvae in a field population of S. frugiperdu; but the only other virus to which S. frugiperda are exposed is the Sf GV, which has not been observed to kill more than 7% (Fuxa, 1982). Populations of S.frugiperdu have developed resistance to methyl parathion (Pitre, 1986); thus, it seems feasible that extensive natural epizootics of nuclear polyhedrosis, such as occur in Louisiana (Fuxa, 1982), and differential NPV susceptibility in S. frugiperdu field populations (Fuxa, 1987; Fuxa et al., 1988) could improve control of the insect by methyl parathion. Many pestiferous insects that are subject to viral epizootics must often be controlled with chemical insecticides; and negatively correlated resistance, which occasionally is observed between chemical in-
CROSS-RESISTANCE
secticides, can provide opportunity for resistance management (Brown and Pal, 1971; Kurtak et al., 1987). However, the negatively correlated resistance in the current research was observed only in the feeding experiments and not by topical application, whereas methyl parathion acts largely as a contact insecticide in field applications . It initially is disturbing to those interested in microbial control to learn that resistance to insect viruses might be a widespread phenomenon (Briese, 1986; Fuxa et al., 1988). However, the reversion to susceptibility (Fuxa and Richter, 1989), the lack of cross-resistance to nonviral mortality agents, and the example of negatively correlated resistance to a chemical are positive features that perhaps can be exploited for resistance management in microbial control. It is clear that further research is necessary to determine whether these phenomena are widespread among different virus-insect systems. ACKNOWLEDGMENTS The authors thank Drs. Jerry B. Graves and Thomas C. Sparks for critically reading the manuscript and for providing helpful suggestions.
REFERENCES BRIESE, D. T. 1986. Insect resistance to baculoviruses. In “The Biology of Baculoviruses,” Vol. 2, “Practical Application for Insect Control” (R. R. Granados and B. A. Federici, Eds.), CRC Press, Boca Raton, FL. BROOKS, G. T. 1976. Penetration and distribution of insecticides. In “Insecticide Biochemistry and Physiology” (C. F. Wilkinson, Ed.), Plenum, New York/London. BROWN, A. W. A., AND PAL, R. 1971. “Insecticide Resistance in Arthropods.” World Health Organi~ation, Geneva. FINNEY, D. J. 1971. “Probit Analysis.” Cambridge Univ. Press, New York. FUXA, J. R. 1982. Prevalence of viral infections in populations of fall armyworm, Spodopreru frugiperda, in southeastern Louisiana. Environ. Entomol., 11, 239-242. FUXA, J. R. 1987. Spodoptera frugiperda susceptibil-
IN RESPONSE TO NPV
277
ity to nuclear polyhedrosis virus isolates with reference to insect migration. Environ. Entomol., 16, 218-223. FUXA, J. R., MITCHELL, F. L., AND RICHTER, A. R. 1988. Resistance of Spodoptera frugiperda [Lep.: Noctuidae] to a nuclear polyhedrosis virus in the field and laboratory. Entomophaga, 33, 55-63. FUXA, J. R., AND RICHTER, A. R. 1989. Reversion of resistance by Spodoptera frugiperda to nuclear polyhedrosis virus. J. Znvertebr. Pathol., 53, 52-56. GREENE, G. L., LEPPLA, N. C., AND DICKERSON, W. A. 1976. Velvetbean caterpillar: a rearing procedure and artificial medium. J. Econ. Entomol., 69, 487-488. HUGHES, P. R., AND WOOD, H. A. 1981. A synchronous peroral technique for the bioassay of insect viruses. J. Znvertebr. Puthol., 37, 154-159. IGNOFFO, C. M., AND ROLJSH, R. T. 1986. Susceptibility of permethrin- and methomyl-resistant strains of Heliothis virescens (Lepidoptera: Noctuidae) to representative species of entomopathogens, J. Econ. Entomol., 19, 334-337. KINSINGER, R. A., AND MCGAUGHEY, W. H. 1979. Susceptibility of populations of Indianmeal moth and almond moth to Bacillus thuringiensis. J. Econ. Entomol., 12, 346-349. KURTAK, D., MEYER, R., OCRAN, M., OUBDRAOGO, M., RENAUD, P., SAWADOGO, R. O., AND TBLI?, B. 1987. Management of insecticide resistance in control of the Simulium damnosum complex by the Onchocerciasis Control Programme, West Africa: Potential use of negative correlation between organophosphate resistance and pyrethroid susceptibility. Med. Vet. Entomol., 1, 137-146. MITCHELL, F. L., AND SMITH, J. W. 1985. Pathology and bioassays of the lesser comstafk borer (Elasmopalpus lignosellus) entomopoxvirus. J. Znvertebr. Pathol., 45, 75-80. PITRE, H. N. 1986. Chemical control of the fall armyworm (Lepidoptera: Noctuidae): An update. Florida Entomol., 69, 570-578. REICHELDERFER, C. F., AND BENTON, C. V. 1974. Some genetic aspects of the resistance of Spodoptera frugiperda to a nuclear polyhedrosis virus. J. Znvertebr. Pathol., 23, 378-382. RICHTER, A. R., AND FUXA, J. R. 1984. Pathogenpathogen and pathogen-insectide interactions in velvetbean caterpillar (Lepidoptera: Noctuidae). J. Econ. Entomol., 11, 1559-1564. STAIRS, G. R. 1980. Comparative infectivity of nonoceluded virions, polyhedra, and virions released from polyhedra for larvae of Galleria mellonella. J. Znvertebr. Pathol., 36, 281-282. WATANABE, H. 1987. The host population. In “Epizootiology of Insect Diseases” (J. R. Fuxa and Y. Tanada, Eds.). Wiley, New York.
