- Email: [email protected]
Evaluation of Steinernema feltiae and S. bibionis (Rhabditida: Steinernematidae) for suppression of Lymantria dispar (Lepidoptera: Lymantriidae) in Pennsylvania, U.S.A.
Agriculture, Ecosystems and Environment, 15 (1986) 1--9 Elsevier Science Publishers B.V., Amsterdam --Printed in The Netherlands
1
E V A L U A T I O N O F S T E I N E R N E M A F E L T I A E A N D S. B I B I O N I S ( R H A B D I T I D A : S T E I N E R N E M A T I D A E ) FOR SUPPRESSION OF L Y M A N T R I A D I S P A R ( L E P I D O P T E R A : L Y M A N T R I I D A E ) IN P E N N S Y L V A N I A , U.S.A.
R.C. REARDON Forest Pest Management, Forest Service, U.S. Department of Agriculture, Morgantown, WV 26505 (U.S.A.) H.K. KAYA
Division of Nematology, University of California, Davis, CA 95616 (U.S.A.) R.A. FUSCO l Pennsylvania Bureau of Forestry, Middletown, PA 1 7057 (U.S.A.) F.B. LEWIS Northeastern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Hamden, CT 60514 (U.S.A.) (Accepted for publication 24 July 1985)
ABSTRACT Reardon, R.C., Kaya, H.K., Fusco, R.A. and Lewis, F.B., 1986. Evaluation of Steinernema feltiae and S. bibionis (Rhabditida: Steinernematidae) for suppression of Lymantria dispar (Lepidoptera: Lymantriidae) in Pennsylvania, U.S.A. Agric. Ecosystems Environ., 15: 1--9.
Lymantria dispar L. larvae exposed to the entomogenous nematodes Steinernema feltiae Filipjev and S. bibionis (Bovien) for /> 16 h in laboratory petri dish tests averaged 93 and 91% mortality, respectively. Nylon pack cloth bands, lined with Pelion fleece or terry cloth, placed around oak tree boles were treated with nematode suspensions in central Pennsylvania. Fifth and 6th stage host larvae collected from beneath these bands after nematode treatment averaged 28% mortality.
INTRODUCTION Chemical (White et al., 1 9 8 1 ) or m i c r o b i a l insecticides ( D u b o i s , 1 9 8 1 ; Lewis and Y e n d o l , 1 9 8 1 ) are used to suppress larval p o p u l a t i o n s o f t h e g y p s y m o t h , L y m a n t r i a dispar L., in f o r e s t e d and residential areas in the n o r t h e a s t e r n U n i t e d States. S p r a y a p p l i c a t i o n o f insecticides is t h e usual m e t h o d f o r individual tree p r o t e c t i o n , a l t h o u g h o f t e n i m p r a c t i c a l f o r h o m e 1Present address: Abbott Laboratories, North Chicago, IL 60064, U.S.A.
0167-8809/86/$03.50
O 1986 Elsevier Science Publishers B.V.
owners and alternate tactics need to be developed. One tactic is to place bands around tree boles, as these bands are ideal resting niches for late stage gypsy moth larvae which feed on foliage in the tree crowns during the night and disperse to the lower bole or in ground litter during the day {Leonard, 1981). Since larvae under the bands are not feeding, one m e t h o d to kill them is the use of steinernematid nematodes which have the ability to seek and actively infect their hosts (Poinar, 1979; Gaugler, 1981). The infective stage of these nematodes enters the host orally or anally and penetrates through the midgut into the hemocoel (Poinar and Himsworth, 1967) or through the spiracular openings into the hemocoelic cavity (Triggianni and Poinar, 1976). This paper reports a study of the effectiveness of two nematode species, Steinernema feltiae Filipjev (= Neoaplectana carpocapsae Weiser} and S. bibionis (Bovien) against gypsy m o t h larvae, under laboratory conditions and in the field, using several types of bands. Steinernema feltiae was used because gypsy moth larvae and pupae were susceptible to this species in petri dish tests (Poinar et al., 1981) and S. bibionis was used because of its effectiveness against the currant borer m o t h (Miller and Bedding, 1982). M AT ER I ALS AND METHODS
Nematode culture Steinernema feltiae, along with its associated bacterium, Xenorhabdus nematophilus (Poinar and Thomas), was cultured monoxenically on polyurethane sponge and harvested according to the m e t h o d developed by Bedding (1981), except that turkey offal was used rather than pork kidney and beef fat. S.bibionis was obtained from Biotechnology Australia Pty Ltd., Roseville, Australia' and shipped by air to California in polyurethane sponge (Bedding, 1984). Both species were stored with continuous aeration at 10°C until used for experiments. Laboratory tests Tests were conducted at the Pennsylvania Bureau of Forestry Laboratory using laboratory-reared gypsy moth larvae. In the first test, five 4th and 5th stage larvae were exposed per replicate to 1000 infective nematodes of S. feltiae or S. bibionis in 1.5 ml of water on a filter paper (9.0 cm, Whatman No. 1) in a 100X 15-mm petri dish for 2, 4, 8, 16, 24 and 48 h. In the second test, 20, 4th and 5th stage larvae were exposed per replicate to 1.0 X 106 infective S. feltiae or S. bibionis in 120 ml of nematode suspension
'Trade names and commercial enterprises or products are mentioned solely for information. N o endorsement by the U.S. Department of Agriculture is implied.
54 5 5
151 6 6
No. of replicates per exposttre time
----
40±7b 2 83+ 10a 0
2
30+llb 6 ± 5b 0
47+6b 57 ± 1 1 a 0
4
35± 17b 3 0 -+ 8 b 0
68+6b 90±6a 0
8
55+- 1 3 b -0
3 ---
12
Mortality after indicated exposure time (h)
----
87-+4a 93+5a 0
16
100 ± 0a 53 ± 12a 0
95±3a 87 +11a 0
24
F._ive 4 t h a n d 5 t h s t a g e l a r v a e e x p o s e d p e r r e p l i c a t e . 2 X = m e a n ; S x = s t a n d a r d e r r o r o f m e a n . M e a n s i n r o w s f o l l o w e d b y t h e s a m e l e t t e r d o n o t d i f f e r s i g n i f i c a n t l y (P ~ 0 . 0 5 ) . 3 indicates no data. 4 Twenty 4th and 5th stage larvae exposed per replicate.
Water (control)
8. feltiae 8. b i b i o n i s
Nylon pack cloth on bole sections
Water (control)
S. feltiae 8. b i b i o n i s
Filter paper in Petri dish
Treatment
1 0 0 +- 0 a 6 0 -+ 6 a 0
36
P e r c e n t m o r t a l i t y (X+ S x ) o f 4 t h a n d 5 t h s t a g e g y p s y m o t h l a r v a e e x p o s e d t o S t e i n e r n e m a feltiae a n d S. b i b i o n i s i n l a b o r a t o r y t e s t s , 1 9 8 3
TABLE I
--
l
± 2a 9 3 + 5a 0 98
48
C,O
sprayed on a Pellon fleece (a polyester padding and quilting material) lining of water-proof 420 denier nylon pack cloth which was wrapped around a cut section (60 cm length × 15 cm diameter} of tree bole. These larvae were confined to the bands using Tanglefoot® applied above and below the bands. Four larvae were removed from the undersides of each band per exposure time, 4, 8, 12, 24 and 36 h. In both laboratory tests, distilled water was sprayed onto the appropriate substrate as controls. After exposure, larvae were transferred individually to 30-ml plastic vials containing high-wheat-germ gypsy m o t h artificial diet (USDA, APHIS). After 7 days, dead larvae were dissected to determine nematode infection. The number of replicates for each treatment is shown in Table I. FIELD TESTS The field portion of the study was conducted on Sideling Hill in Buchanan State Forest in central Pennsylvania. The forest type was oak--red maple with chestnut oak, Quercus prinus L., comprising 70% of the dominant trees.
1983 G y p s y moth populations were peaking with an average of 3000 egg masses per ha. A total of 35 chestnut oak or black oak, Q. velutina Lain., were banded at breast height (approximately 1.5 m) and treatments randomly assigned. Fifteen trees were treated with Tanglefoot® applied above and below each band, 0.3 m wide, to confine the larvae beneath the bands. Of these 15 trees, 10 had the nylon pack cloth band lined with Pellon fleece and treated with a nematode suspension and five were untreated controls. For the remaining 20 trees, larvae were not restricted to the bands: 20 had nylon pack cloth bands (15 with the Pellon fleece lining treated and five untreated controls). Bands were treated with the nematode suspension on 23 June (1800-2000 h). During treatment, the ambient temperature averaged 25°C and relative humidity 40%. An average of 568 ml of S. feltiae suspension (1 × 106 nematodes) was applied to each treatment tree band (~ 538 nematodes cm -2) using a hand-pump sprayer. For the trees with Tanglefoot®, a random subsample of five larvae maximum was collected from each band 6, 12, 36 and 120 h after treatment. For the trees without Tanglefoot®, a random subsample of 10 larvae maximum was collected from each band at 60 and 120 h after treatment. Larvae were placed individually in 30-ml plastic vials containing artificial diet and checked for mortality 7--10 days after collection. Dead and moribund larvae were examined for nematode and gypsy m o t h nucleopolyhedrosis virus (NPV) infection.
1984
Gypsy m o t h egg mass density was lower in 1984, averaging 1500 ha -1. A total of 60 chestnut oaks were banded with nylon pack cloth lined with terry cloth. Fifty bands were treated with nematode suspension, five treated with water {treated controls) and five were untreated controls. Treatments were randomly assigned. A sub-sample of the bands was treated with nematode suspensions on 22 June {Trial 1), 23 June (Trial 2) and 2 July {Trial 3). The bands for Trial 2 were treated at 0700--0830 h (the ambient temperature averaged 20°C and relative humidity 90%) and the bands for Trial 1 and 3 at 1800--2000 h (25°C and 80% humidity). An average of 284 ml of S. feltiae or S. bibionis (2 × 106 nematodes) was applied to the lining o f each treatment tree band using a hand-pump sprayer. A random subsample of 10 larvae m a x i m u m was collected from each band at selected hours after treatment. Larvae were handled as in 1983. All data was analyzed by using one-way analysis of variance (ANOVA) to detect differences among means and Scheffe's test to detect differences between means (at the a = 0.05 level). RESULTS L a b o r a t o r y tests
Late stage gypsy m o t h larvae exposed to S. feltiae and S. bibionis on filter paper in petri dishes for ~> 16 h averaged 93% and 91% mortality, respectively {Table I). The percent mortalities of 4th and 5th stage gypsy moth larvae for the various exposure times were analyzed by one-way ANOVA. Differences were significant among treatment exposure times and checks (S. feltiae - - F = 44.41, P < 0.01; S. bibionis - - F = 12.34, P < 0.01). The percent mortalities for various exposure times were compared separately by using Scheffe's test. For S. bibionis, the percent mortalities for each exposure time differed from the check, b u t not from each other. For S. feltiae, the percent mortalities for each exposure time differed from the check and some from each other. In general, gypsy m o t h larvae were more susceptible to S. feltiae when exposed for /> 16 h. In the second test, differences were significant among treatment exposure times and checks (S. feltiae - - F = 8.47, P < 0.01; S. bibionis - - F = 9.67, P < 0.01). For S. feltiae and S. bibionis, the percent mortalities of host larvae for the 24- and 36-h exposure times differed from the check and other exposure times. Mortality of gypsy moth larvae exposed to nematodes for 24 and 36 h under nylon pack cloth bands wrapped around bole sections averaged 57% for S. bibionis and 100% forS. feltiae.
6
Field tests 1983 When 5th and 6th stage gypsy m o t h larvae were restricted to nylon pack cloth bands for 6, 12 and 36 h, an average of 39% mortality attributable to S. feltiae occurred (Table II). When host larvae were exposed for 120 h, the percent mortality for both restricted and non-restricted larvae was less than for shorter exposure times. The percent mortality of gypsy m o t h larvae attributable to S. feltiae was lower in the field than in the laboratory. T A B L E II M o r t a l i t y o f 5th a n d 6 t h stage g y p s y m o t h larvae e x p o s e d t o Steinernema feltiae in field tests in P e n n s y l v a n i a , 19 8 3 Treatment
N o . of bands
Larval exposure (h)
Total n u m b e r of larvae
Cause of d e a t h (No. l a r v a e ) NPV a
Nematode
Mortality attributable to n e m a t o d e s (%)
Larvae r e s t r i c t e d to bands b Nylon p a c k c l o t h band~reated
Untreated controls
10
6 12 36 120
20 30 40 50
4 8 9 16
8 12 15 6
40 40 38 12
5
6 12 36 120
10 10 10 10
2 5 5 8
0 0 0 0
0 0 0 0
15
60 120
135 25
66 12
13 4
10 16
5
60 120
30 25
4 5
0 0
0 0
Larvae not restricted to bands Nylon pack cloth band-treated
Untreated controls
aNPV = gypsy m o t h nucleopolyhedrogis virus. b L a r v a e r e s t r i c t e d t o b a n d s using T a n g l e f o o t (~ a p p l i e d a b o v e a n d b e l o w t h e b a n d s .
1984 Based on field tests conducted in 1983, the nematode concentration was increased to 2 × 106 per band and a more absorbent lining (terry cloth) was utilized under the nylon pack cloth. Also, host larvae were not confined to the bands. For the bands and host larvae under the bands treated in the morning (Trials 1 and 3), a m a x i m u m of 74% mortality of 5th and 6th stage larvae was recorded for 6 h exposure and a minimum of 11% for 72 h exposure to S. feltiae (Table III). These percent mortalities recorded for non-restricted larvae in 1984 were similar, except for the 6-h exposure, to those recorded for restricted larvae in 1983. For S. bibionis, a m a x i m u m of
7 T A B L E III M o r t a l i t y o f 5 t h a n d 6 t h stage g y p s y m o t h I:,rvae e x p o s e d t o Steinernema feltiae a n d
S. bibionis in field t e s t s in P e n n s y l v a n i a , 1 9 8 4 a Treatment
No. o f bands
Larval exposure (h)
Total number of larvae
Cause o f d e a t h (No. larvae) NPV
Nematode
Mortality attributable to n e m a t o d e s (%)
Trials l a n d 3
S. feltiae
10
6 10 24 48 72
46 52 50 35 28
6 14 32 18 24
34 24 17 5 3
74 46 34 14 11
S. bibionis
10
6 10 24 48 72
40 46 83 31 91
19 24 55 16 85
6 5 28 0 2
15 11 34 0 2
Water
5
6 10 48
10 10 20
4 4 10
0 0 0
0 0 0
Check
5
6 10 24 48
20 12 10 17
1 3 6 2
0 0 0 0
0 0 0 0
S. feltiae
10
1 10 36 60 120
66 61 44 50 73
22 5 13 21 35
31 45 24 2 4
47 74 55 4 5
S. bibionis
10
10 36
19 25
2 10
14 0
74 0
Water
5
10 36
24 13
14 10
0 0
0 0
Check
5
10 36
20 10
8 4
0 0
0 0
Trial 2
aEach species of nematode was applied to nylon pack cloth bands lined with terry cloth.
34% mortality was recorded for host larvae exposed for 24 h and 2% mortality for 72 h {Table III). For the bands and host larvae under the bands treated in the evening {Trial 2), a m a x i m u m of 74% mortality of host larvae was recorded for S. feltiae and S. bibionis when exposed for 10 h. These mortality percentages for 10 h exposure were higher than those for larvae collected from beneath the bands treated in the morning for a comparable exposure time. DISCUSSION
Fourth and 5th stage gypsy m o t h larvae are susceptible to S. feltiae and S. bibionis when exposed in petri dishes and under nylon pack cloth bands in laboratory tests. Both nematode species were equally infectious to host larvae in petri dish tests (/> 16 h exposure for /> 90% mortality, while S. feltiae was more infectious under n y l o n bands. Apparently, the infective juveniles of S. bibionis were more sensitive to moisture levels than S.feltiae for exposure times/> 24 h. In field tests, a m a x i m u m of 40% mortality of host larvae attributable to S. feltiae was recorded in 1983. These host larvae were exposed to 1 × 106 nematodes per nylon band lined with Pellon fleece. In 1984, a m a x i m u m of 74% mortality of host larvae was recorded for ~< 10 h exposure to S. feltiae or S. bibionis. These host larvae were exposed to 2X 106 nematodes per nylon band lined with terry cloth. In general, the percent mortalities for host larvae attributable to S. bibionis were lower than those for S. feltiae. Although the nematodes are much less infective to gypsy m o t h larvae under field conditions, the m a x i m u m potential of their use in the biological control of this insect is dependent upon a number of factors. First, the development of a band that retains higher moisture levels allowing nematode survival and which is still attractive to gypsy m o t h larvae. Such a band could be a plastic sheet lined with polyurethane sponge. Second, only a small portion of the gypsy m o t h larvae on each tree were utilizing the bands. Although these larvae had a high incidence of mortality attributable to nematodes and NPV, the trees were at least 40% defoliated. Consequently, the optimal gypsy m o t h density or situation in which gypsy moth larvae utilize the bands and noticeable defoliation does not occur, must be determined. Third, a nematode strain or species more efficacious to gypsy m o t h larvae must be found. Molyneux et al. {1983) and Silverman et al. {1982) demonstrated that certain strains of steinernematids were more efficacious than others against blow flies and fleas, respectively. Fourth, the NPV and n e m a t o d e interaction requires more research to determine whether NPV infection prevents n e m a t o d e infection or n e m a t o d e establishment. An area that requires more research is the use of the nematode against gypsy m o t h pupae. Preliminary studies show t h a t a small percentage of the
pupae were susceptible to the nematode in the field. Since pupae remain in sites for 5 or more days, the possibility of enhancing nematode infection is good. A number of problems including nematode survival, efficaciou~ nematode strains, application techniques and nematode infection processes must be overcome for successful use. ACKNOWLEDGEMENTS
We thank Lynda Liptrap, Brad Onken and Mike Blumenthal for field assistance. This work was funded in part by the Northeastern Forest Experiment Station, Forest Service, USDA, Cooperative Agreement 23-869.
REFERENCES Bedding, R.A., 1981. Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica, 27: 109--114. Bedding, R.A., 1984. Large scale production, storage and transport of the insect parasitic nematodes Neoaplectana spp. and Heterorhabditis spp. Ann. Appl. Biol., 104: 117-120. Dubois, N.R., 1981. Microbials. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 445--453. Gaugler, R., 1981. Biological control potential of neoapleetanid nematodes. J. Nematol., 13: 241--249. Leonard, D.E., 1981. Bioecology of the gypsy moth. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bun., 1584, pp. 9--29. Lewis, F.B. and Yendol, W.G:, 1981. Microbials. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 503--512. Miller, L.A. and Bedding, R.A., 1982. Field testing of the insect parasitic nematode Neoaplectana bibionis (Nematoda: Steinernematidae) against currant borer moth, Synanthedon tipuliformis, (Lepidoptera: Sesiidae) in black currants. Entomophaga, 27: 109--114. Molyneux, A.S., Bedding, R.A. and Akhurst, R.J., 1983. Susceptibility of larvae of the sheep blowfly Lucilia cuprina to various Heterorhabditis spp., Neoaplectana spp., and an underscribed steinernematid (Nematoda). J. Invertebr. Pathol., 42: 1--7. Poinar, G.O., Jr., 1979. Nematodes for Biological (~ontrol of Insects. CRC Press, Boca Raton, FL, 277 pp. Poinar, G.O., Jr. and Himsworth, P.T., 1967. Neoaplectana parasitism of larvae of the greater wax moth, Galleria mellonella. J. Invertebr. Pathol., 9: 241--246. Poinar, G.O., Jr., Shapiro, M. and Lindegren, J.E., 1981. Susceptibility of the gypsy moth (Lymantria dispar) to the parasitic nematode, Neoaplectana carpocapsae. IRCS Med. Sci., 9: 985. Silverman, J., Platzer, E.G. and Rust, M.K., 1982. Infection of the rat flea Ctenocephalides felis (Bouche) by Neoaplectana carpocapsae Weiser. J. Nematol., 14: 394--397. Triggianni, O. and Poinar, G.O., Jr., 1976. Infection of adult Lepidoptera by Neoaplectana carpocapsae (Nematoda). J. Invertebr. Pathol., 27: 413--414. White, W.B., McLane, W.H. and Schneeberger N.F., 1981. Pesticides. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 423--442.
1
E V A L U A T I O N O F S T E I N E R N E M A F E L T I A E A N D S. B I B I O N I S ( R H A B D I T I D A : S T E I N E R N E M A T I D A E ) FOR SUPPRESSION OF L Y M A N T R I A D I S P A R ( L E P I D O P T E R A : L Y M A N T R I I D A E ) IN P E N N S Y L V A N I A , U.S.A.
R.C. REARDON Forest Pest Management, Forest Service, U.S. Department of Agriculture, Morgantown, WV 26505 (U.S.A.) H.K. KAYA
Division of Nematology, University of California, Davis, CA 95616 (U.S.A.) R.A. FUSCO l Pennsylvania Bureau of Forestry, Middletown, PA 1 7057 (U.S.A.) F.B. LEWIS Northeastern Forest Experiment Station, Forest Service, U.S. Department of Agriculture, Hamden, CT 60514 (U.S.A.) (Accepted for publication 24 July 1985)
ABSTRACT Reardon, R.C., Kaya, H.K., Fusco, R.A. and Lewis, F.B., 1986. Evaluation of Steinernema feltiae and S. bibionis (Rhabditida: Steinernematidae) for suppression of Lymantria dispar (Lepidoptera: Lymantriidae) in Pennsylvania, U.S.A. Agric. Ecosystems Environ., 15: 1--9.
Lymantria dispar L. larvae exposed to the entomogenous nematodes Steinernema feltiae Filipjev and S. bibionis (Bovien) for /> 16 h in laboratory petri dish tests averaged 93 and 91% mortality, respectively. Nylon pack cloth bands, lined with Pelion fleece or terry cloth, placed around oak tree boles were treated with nematode suspensions in central Pennsylvania. Fifth and 6th stage host larvae collected from beneath these bands after nematode treatment averaged 28% mortality.
INTRODUCTION Chemical (White et al., 1 9 8 1 ) or m i c r o b i a l insecticides ( D u b o i s , 1 9 8 1 ; Lewis and Y e n d o l , 1 9 8 1 ) are used to suppress larval p o p u l a t i o n s o f t h e g y p s y m o t h , L y m a n t r i a dispar L., in f o r e s t e d and residential areas in the n o r t h e a s t e r n U n i t e d States. S p r a y a p p l i c a t i o n o f insecticides is t h e usual m e t h o d f o r individual tree p r o t e c t i o n , a l t h o u g h o f t e n i m p r a c t i c a l f o r h o m e 1Present address: Abbott Laboratories, North Chicago, IL 60064, U.S.A.
0167-8809/86/$03.50
O 1986 Elsevier Science Publishers B.V.
owners and alternate tactics need to be developed. One tactic is to place bands around tree boles, as these bands are ideal resting niches for late stage gypsy moth larvae which feed on foliage in the tree crowns during the night and disperse to the lower bole or in ground litter during the day {Leonard, 1981). Since larvae under the bands are not feeding, one m e t h o d to kill them is the use of steinernematid nematodes which have the ability to seek and actively infect their hosts (Poinar, 1979; Gaugler, 1981). The infective stage of these nematodes enters the host orally or anally and penetrates through the midgut into the hemocoel (Poinar and Himsworth, 1967) or through the spiracular openings into the hemocoelic cavity (Triggianni and Poinar, 1976). This paper reports a study of the effectiveness of two nematode species, Steinernema feltiae Filipjev (= Neoaplectana carpocapsae Weiser} and S. bibionis (Bovien) against gypsy m o t h larvae, under laboratory conditions and in the field, using several types of bands. Steinernema feltiae was used because gypsy moth larvae and pupae were susceptible to this species in petri dish tests (Poinar et al., 1981) and S. bibionis was used because of its effectiveness against the currant borer m o t h (Miller and Bedding, 1982). M AT ER I ALS AND METHODS
Nematode culture Steinernema feltiae, along with its associated bacterium, Xenorhabdus nematophilus (Poinar and Thomas), was cultured monoxenically on polyurethane sponge and harvested according to the m e t h o d developed by Bedding (1981), except that turkey offal was used rather than pork kidney and beef fat. S.bibionis was obtained from Biotechnology Australia Pty Ltd., Roseville, Australia' and shipped by air to California in polyurethane sponge (Bedding, 1984). Both species were stored with continuous aeration at 10°C until used for experiments. Laboratory tests Tests were conducted at the Pennsylvania Bureau of Forestry Laboratory using laboratory-reared gypsy moth larvae. In the first test, five 4th and 5th stage larvae were exposed per replicate to 1000 infective nematodes of S. feltiae or S. bibionis in 1.5 ml of water on a filter paper (9.0 cm, Whatman No. 1) in a 100X 15-mm petri dish for 2, 4, 8, 16, 24 and 48 h. In the second test, 20, 4th and 5th stage larvae were exposed per replicate to 1.0 X 106 infective S. feltiae or S. bibionis in 120 ml of nematode suspension
'Trade names and commercial enterprises or products are mentioned solely for information. N o endorsement by the U.S. Department of Agriculture is implied.
54 5 5
151 6 6
No. of replicates per exposttre time
----
40±7b 2 83+ 10a 0
2
30+llb 6 ± 5b 0
47+6b 57 ± 1 1 a 0
4
35± 17b 3 0 -+ 8 b 0
68+6b 90±6a 0
8
55+- 1 3 b -0
3 ---
12
Mortality after indicated exposure time (h)
----
87-+4a 93+5a 0
16
100 ± 0a 53 ± 12a 0
95±3a 87 +11a 0
24
F._ive 4 t h a n d 5 t h s t a g e l a r v a e e x p o s e d p e r r e p l i c a t e . 2 X = m e a n ; S x = s t a n d a r d e r r o r o f m e a n . M e a n s i n r o w s f o l l o w e d b y t h e s a m e l e t t e r d o n o t d i f f e r s i g n i f i c a n t l y (P ~ 0 . 0 5 ) . 3 indicates no data. 4 Twenty 4th and 5th stage larvae exposed per replicate.
Water (control)
8. feltiae 8. b i b i o n i s
Nylon pack cloth on bole sections
Water (control)
S. feltiae 8. b i b i o n i s
Filter paper in Petri dish
Treatment
1 0 0 +- 0 a 6 0 -+ 6 a 0
36
P e r c e n t m o r t a l i t y (X+ S x ) o f 4 t h a n d 5 t h s t a g e g y p s y m o t h l a r v a e e x p o s e d t o S t e i n e r n e m a feltiae a n d S. b i b i o n i s i n l a b o r a t o r y t e s t s , 1 9 8 3
TABLE I
--
l
± 2a 9 3 + 5a 0 98
48
C,O
sprayed on a Pellon fleece (a polyester padding and quilting material) lining of water-proof 420 denier nylon pack cloth which was wrapped around a cut section (60 cm length × 15 cm diameter} of tree bole. These larvae were confined to the bands using Tanglefoot® applied above and below the bands. Four larvae were removed from the undersides of each band per exposure time, 4, 8, 12, 24 and 36 h. In both laboratory tests, distilled water was sprayed onto the appropriate substrate as controls. After exposure, larvae were transferred individually to 30-ml plastic vials containing high-wheat-germ gypsy m o t h artificial diet (USDA, APHIS). After 7 days, dead larvae were dissected to determine nematode infection. The number of replicates for each treatment is shown in Table I. FIELD TESTS The field portion of the study was conducted on Sideling Hill in Buchanan State Forest in central Pennsylvania. The forest type was oak--red maple with chestnut oak, Quercus prinus L., comprising 70% of the dominant trees.
1983 G y p s y moth populations were peaking with an average of 3000 egg masses per ha. A total of 35 chestnut oak or black oak, Q. velutina Lain., were banded at breast height (approximately 1.5 m) and treatments randomly assigned. Fifteen trees were treated with Tanglefoot® applied above and below each band, 0.3 m wide, to confine the larvae beneath the bands. Of these 15 trees, 10 had the nylon pack cloth band lined with Pellon fleece and treated with a nematode suspension and five were untreated controls. For the remaining 20 trees, larvae were not restricted to the bands: 20 had nylon pack cloth bands (15 with the Pellon fleece lining treated and five untreated controls). Bands were treated with the nematode suspension on 23 June (1800-2000 h). During treatment, the ambient temperature averaged 25°C and relative humidity 40%. An average of 568 ml of S. feltiae suspension (1 × 106 nematodes) was applied to each treatment tree band (~ 538 nematodes cm -2) using a hand-pump sprayer. For the trees with Tanglefoot®, a random subsample of five larvae maximum was collected from each band 6, 12, 36 and 120 h after treatment. For the trees without Tanglefoot®, a random subsample of 10 larvae maximum was collected from each band at 60 and 120 h after treatment. Larvae were placed individually in 30-ml plastic vials containing artificial diet and checked for mortality 7--10 days after collection. Dead and moribund larvae were examined for nematode and gypsy m o t h nucleopolyhedrosis virus (NPV) infection.
1984
Gypsy m o t h egg mass density was lower in 1984, averaging 1500 ha -1. A total of 60 chestnut oaks were banded with nylon pack cloth lined with terry cloth. Fifty bands were treated with nematode suspension, five treated with water {treated controls) and five were untreated controls. Treatments were randomly assigned. A sub-sample of the bands was treated with nematode suspensions on 22 June {Trial 1), 23 June (Trial 2) and 2 July {Trial 3). The bands for Trial 2 were treated at 0700--0830 h (the ambient temperature averaged 20°C and relative humidity 90%) and the bands for Trial 1 and 3 at 1800--2000 h (25°C and 80% humidity). An average of 284 ml of S. feltiae or S. bibionis (2 × 106 nematodes) was applied to the lining o f each treatment tree band using a hand-pump sprayer. A random subsample of 10 larvae m a x i m u m was collected from each band at selected hours after treatment. Larvae were handled as in 1983. All data was analyzed by using one-way analysis of variance (ANOVA) to detect differences among means and Scheffe's test to detect differences between means (at the a = 0.05 level). RESULTS L a b o r a t o r y tests
Late stage gypsy m o t h larvae exposed to S. feltiae and S. bibionis on filter paper in petri dishes for ~> 16 h averaged 93% and 91% mortality, respectively {Table I). The percent mortalities of 4th and 5th stage gypsy moth larvae for the various exposure times were analyzed by one-way ANOVA. Differences were significant among treatment exposure times and checks (S. feltiae - - F = 44.41, P < 0.01; S. bibionis - - F = 12.34, P < 0.01). The percent mortalities for various exposure times were compared separately by using Scheffe's test. For S. bibionis, the percent mortalities for each exposure time differed from the check, b u t not from each other. For S. feltiae, the percent mortalities for each exposure time differed from the check and some from each other. In general, gypsy m o t h larvae were more susceptible to S. feltiae when exposed for /> 16 h. In the second test, differences were significant among treatment exposure times and checks (S. feltiae - - F = 8.47, P < 0.01; S. bibionis - - F = 9.67, P < 0.01). For S. feltiae and S. bibionis, the percent mortalities of host larvae for the 24- and 36-h exposure times differed from the check and other exposure times. Mortality of gypsy moth larvae exposed to nematodes for 24 and 36 h under nylon pack cloth bands wrapped around bole sections averaged 57% for S. bibionis and 100% forS. feltiae.
6
Field tests 1983 When 5th and 6th stage gypsy m o t h larvae were restricted to nylon pack cloth bands for 6, 12 and 36 h, an average of 39% mortality attributable to S. feltiae occurred (Table II). When host larvae were exposed for 120 h, the percent mortality for both restricted and non-restricted larvae was less than for shorter exposure times. The percent mortality of gypsy m o t h larvae attributable to S. feltiae was lower in the field than in the laboratory. T A B L E II M o r t a l i t y o f 5th a n d 6 t h stage g y p s y m o t h larvae e x p o s e d t o Steinernema feltiae in field tests in P e n n s y l v a n i a , 19 8 3 Treatment
N o . of bands
Larval exposure (h)
Total n u m b e r of larvae
Cause of d e a t h (No. l a r v a e ) NPV a
Nematode
Mortality attributable to n e m a t o d e s (%)
Larvae r e s t r i c t e d to bands b Nylon p a c k c l o t h band~reated
Untreated controls
10
6 12 36 120
20 30 40 50
4 8 9 16
8 12 15 6
40 40 38 12
5
6 12 36 120
10 10 10 10
2 5 5 8
0 0 0 0
0 0 0 0
15
60 120
135 25
66 12
13 4
10 16
5
60 120
30 25
4 5
0 0
0 0
Larvae not restricted to bands Nylon pack cloth band-treated
Untreated controls
aNPV = gypsy m o t h nucleopolyhedrogis virus. b L a r v a e r e s t r i c t e d t o b a n d s using T a n g l e f o o t (~ a p p l i e d a b o v e a n d b e l o w t h e b a n d s .
1984 Based on field tests conducted in 1983, the nematode concentration was increased to 2 × 106 per band and a more absorbent lining (terry cloth) was utilized under the nylon pack cloth. Also, host larvae were not confined to the bands. For the bands and host larvae under the bands treated in the morning (Trials 1 and 3), a m a x i m u m of 74% mortality of 5th and 6th stage larvae was recorded for 6 h exposure and a minimum of 11% for 72 h exposure to S. feltiae (Table III). These percent mortalities recorded for non-restricted larvae in 1984 were similar, except for the 6-h exposure, to those recorded for restricted larvae in 1983. For S. bibionis, a m a x i m u m of
7 T A B L E III M o r t a l i t y o f 5 t h a n d 6 t h stage g y p s y m o t h I:,rvae e x p o s e d t o Steinernema feltiae a n d
S. bibionis in field t e s t s in P e n n s y l v a n i a , 1 9 8 4 a Treatment
No. o f bands
Larval exposure (h)
Total number of larvae
Cause o f d e a t h (No. larvae) NPV
Nematode
Mortality attributable to n e m a t o d e s (%)
Trials l a n d 3
S. feltiae
10
6 10 24 48 72
46 52 50 35 28
6 14 32 18 24
34 24 17 5 3
74 46 34 14 11
S. bibionis
10
6 10 24 48 72
40 46 83 31 91
19 24 55 16 85
6 5 28 0 2
15 11 34 0 2
Water
5
6 10 48
10 10 20
4 4 10
0 0 0
0 0 0
Check
5
6 10 24 48
20 12 10 17
1 3 6 2
0 0 0 0
0 0 0 0
S. feltiae
10
1 10 36 60 120
66 61 44 50 73
22 5 13 21 35
31 45 24 2 4
47 74 55 4 5
S. bibionis
10
10 36
19 25
2 10
14 0
74 0
Water
5
10 36
24 13
14 10
0 0
0 0
Check
5
10 36
20 10
8 4
0 0
0 0
Trial 2
aEach species of nematode was applied to nylon pack cloth bands lined with terry cloth.
34% mortality was recorded for host larvae exposed for 24 h and 2% mortality for 72 h {Table III). For the bands and host larvae under the bands treated in the evening {Trial 2), a m a x i m u m of 74% mortality of host larvae was recorded for S. feltiae and S. bibionis when exposed for 10 h. These mortality percentages for 10 h exposure were higher than those for larvae collected from beneath the bands treated in the morning for a comparable exposure time. DISCUSSION
Fourth and 5th stage gypsy m o t h larvae are susceptible to S. feltiae and S. bibionis when exposed in petri dishes and under nylon pack cloth bands in laboratory tests. Both nematode species were equally infectious to host larvae in petri dish tests (/> 16 h exposure for /> 90% mortality, while S. feltiae was more infectious under n y l o n bands. Apparently, the infective juveniles of S. bibionis were more sensitive to moisture levels than S.feltiae for exposure times/> 24 h. In field tests, a m a x i m u m of 40% mortality of host larvae attributable to S. feltiae was recorded in 1983. These host larvae were exposed to 1 × 106 nematodes per nylon band lined with Pellon fleece. In 1984, a m a x i m u m of 74% mortality of host larvae was recorded for ~< 10 h exposure to S. feltiae or S. bibionis. These host larvae were exposed to 2X 106 nematodes per nylon band lined with terry cloth. In general, the percent mortalities for host larvae attributable to S. bibionis were lower than those for S. feltiae. Although the nematodes are much less infective to gypsy m o t h larvae under field conditions, the m a x i m u m potential of their use in the biological control of this insect is dependent upon a number of factors. First, the development of a band that retains higher moisture levels allowing nematode survival and which is still attractive to gypsy m o t h larvae. Such a band could be a plastic sheet lined with polyurethane sponge. Second, only a small portion of the gypsy m o t h larvae on each tree were utilizing the bands. Although these larvae had a high incidence of mortality attributable to nematodes and NPV, the trees were at least 40% defoliated. Consequently, the optimal gypsy m o t h density or situation in which gypsy moth larvae utilize the bands and noticeable defoliation does not occur, must be determined. Third, a nematode strain or species more efficacious to gypsy m o t h larvae must be found. Molyneux et al. {1983) and Silverman et al. {1982) demonstrated that certain strains of steinernematids were more efficacious than others against blow flies and fleas, respectively. Fourth, the NPV and n e m a t o d e interaction requires more research to determine whether NPV infection prevents n e m a t o d e infection or n e m a t o d e establishment. An area that requires more research is the use of the nematode against gypsy m o t h pupae. Preliminary studies show t h a t a small percentage of the
pupae were susceptible to the nematode in the field. Since pupae remain in sites for 5 or more days, the possibility of enhancing nematode infection is good. A number of problems including nematode survival, efficaciou~ nematode strains, application techniques and nematode infection processes must be overcome for successful use. ACKNOWLEDGEMENTS
We thank Lynda Liptrap, Brad Onken and Mike Blumenthal for field assistance. This work was funded in part by the Northeastern Forest Experiment Station, Forest Service, USDA, Cooperative Agreement 23-869.
REFERENCES Bedding, R.A., 1981. Low cost in vitro mass production of Neoaplectana and Heterorhabditis species (Nematoda) for field control of insect pests. Nematologica, 27: 109--114. Bedding, R.A., 1984. Large scale production, storage and transport of the insect parasitic nematodes Neoaplectana spp. and Heterorhabditis spp. Ann. Appl. Biol., 104: 117-120. Dubois, N.R., 1981. Microbials. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 445--453. Gaugler, R., 1981. Biological control potential of neoapleetanid nematodes. J. Nematol., 13: 241--249. Leonard, D.E., 1981. Bioecology of the gypsy moth. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bun., 1584, pp. 9--29. Lewis, F.B. and Yendol, W.G:, 1981. Microbials. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 503--512. Miller, L.A. and Bedding, R.A., 1982. Field testing of the insect parasitic nematode Neoaplectana bibionis (Nematoda: Steinernematidae) against currant borer moth, Synanthedon tipuliformis, (Lepidoptera: Sesiidae) in black currants. Entomophaga, 27: 109--114. Molyneux, A.S., Bedding, R.A. and Akhurst, R.J., 1983. Susceptibility of larvae of the sheep blowfly Lucilia cuprina to various Heterorhabditis spp., Neoaplectana spp., and an underscribed steinernematid (Nematoda). J. Invertebr. Pathol., 42: 1--7. Poinar, G.O., Jr., 1979. Nematodes for Biological (~ontrol of Insects. CRC Press, Boca Raton, FL, 277 pp. Poinar, G.O., Jr. and Himsworth, P.T., 1967. Neoaplectana parasitism of larvae of the greater wax moth, Galleria mellonella. J. Invertebr. Pathol., 9: 241--246. Poinar, G.O., Jr., Shapiro, M. and Lindegren, J.E., 1981. Susceptibility of the gypsy moth (Lymantria dispar) to the parasitic nematode, Neoaplectana carpocapsae. IRCS Med. Sci., 9: 985. Silverman, J., Platzer, E.G. and Rust, M.K., 1982. Infection of the rat flea Ctenocephalides felis (Bouche) by Neoaplectana carpocapsae Weiser. J. Nematol., 14: 394--397. Triggianni, O. and Poinar, G.O., Jr., 1976. Infection of adult Lepidoptera by Neoaplectana carpocapsae (Nematoda). J. Invertebr. Pathol., 27: 413--414. White, W.B., McLane, W.H. and Schneeberger N.F., 1981. Pesticides. In: M. McManus and C. Doane (Editors), The Gypsy Moth: Research toward Integrated Pest Management. USDA Tech. Bull., 1584, pp. 423--442.