Selection for Pesticide Resistance in Trioxys Pallidus

TITLE: Selecting for Pesticide Resistance in Trioxys pallidus

Principal Investigator: Marjorie A. Hoy, Department of Entomological Sciences, 201 Wellman Hall, University of Calfiomia, Berkeley 94720 (415) 642- 3989 December IS, 1988

Abstract: The Guthion-resistant strain of walnut aphid parasite, Trio x y s p aII i d us. was mass reared in the greenhouse on potted walnut trees and released into five commercial walnut blocks near Colusa, Stockton, Modesto, and Hanford. Parasites successfully established in four of the five sites, surviving on field rates of Guthion and Supracide. The Guthion-resistant strain persisted in the release orchards throughout the growing season. Samples of parasites from the release sites in October indicated the parasites had maintained their Guthion resistance. In addition, Guthion-resistant parasites were collected from walnut trees in nearby non-release sites, which indicates the Guthion-resistant strain has spread from the release sites. Such natural dispersal will make implementation easier. The Guthion-resistant and a wild strain of T. pallidus were tested with field-collected foliage treated with field rates of Supracide, Zolone, Thiodan, and Lorsban using a clip cage bioassay. The results indicate that the laboratory-selected Guthion-resistant strain is cross resistant to all these pesticides. This is very useful information, as it will help in the establishment of the resistant strain in the field; nearly any pesticide applied in walnut IPM will assist in selecting for the Guthion-resistant strain of T. pall idus. It also makes the Guthion-resistant strain more valuable; even if the grower doesn't use Guthion, this parasite survives better than the wild strain on the major pesticides used in walnut IPM, which should reduce the likelihood of secondary outbreaks of walnut aphids after pesticide use. Tests to determine mode of inheritance of the Guthion resistance are now underway. Complete concentration mortality lines of the pure colonies will be obtained at that time.

Objectives: The objectives for the funding interval were: 1) Complete selection for Guthion resistance with the Guthion-resistant colony of I.. p aII idu s and develop concentration/mortality lines for Resistant, Base and Yolo colonies to document the resistance levels achieved. 2) Test Lorsban, Supracide, Thiodan, and Zolone to determine whether the Guthion-resistant strain of T. P aIii du s exhibits cross resistance to these pesticides. 3) Conduct genetic analyses to determine mode of inheritance of the Guthion resistance. 4) Conduct field evaluations of the Guthion-resistant strain in commercial walnut orchards. Evaluate efficacy overwintering ability and dispersal rates.

Results: Objectives 2) and 4) were completed. Objectives 1) and 3) are being conducted currently.

The results for Objective 2) are presented in the ID:.afl manuscript below:

Toxicity of Pesticides Used In Walnuts to a Wild and Laboratory- Selected Azlnphosmethyl-Reslstant Strain of Trloxvs pallid us (Hymenoptera: Aphldlldae)

80 ------MARJORIEA HOYANDFRANCESE.CAVE J. Econ. Entomol. ABSTRACT The effect of azinphosmethyl (Guthion), chlorpyrifos (Supracide), endosulfan (Thiodan), methidathion (Lorsban), or phosalone (Zolone) on a wild and a laboratory-selected strain of Trioxys pallid us was tested using a clip cage and field-treated walnut foliage. Mortality of adults was assayed after 4, 8, 18, 24, 48, and 72 hrs at 27 .:t.1.50 C. Residues ranged from one to 35 days old. One-day-old residues of chlorpyrifos were the most toxic to the wild colony, while azinphosmethyl, methidathion, phosalone, and endosulfan were less toxic, in this order. Azinphosmethyl residues remained toxic to both colonies longest. Methidathion residues remained toxic to the wild colony after 28 days, but the Selected strain survived on 14-day-oid residues. Residues of phosalone and endosulfan exhibited low toxicity to both strains after 14 days. The azinphosmethyl-selected laboratory strain of I. pallidus survived all pesticide residues, except the 14'-day-old endosulfan residues, better than the wild strain, suggesting that cross resistances were developed through artificial selection with azinphosmethyl. The implications for walnut pest management and orchard management of the new azinphosmethyl-resistant strain of I. pallidus are discussed.

THE WALNUT APHID, Chromaphis juglandicola Kaltenbach, is probably the most important insect attacking Persian or English walnut (,Juglans mgia L.) in California, causing as much as 50 percent reduction in the developing crop if damage occurs in the spring (Michelbacher & Ortega 1958). Midsummer walnut aphid populations in excess of 15 per leaflet may reduce quality of walnuts (Riedl et al. 1979, Sibbett et al. 1981, UC/IPM 1982). Trioxvs pallid us Haliday (Hymenoptera: Aphidiidae) was introduced Into California from France in 1959 and subsequently provided control of walnut aphid in the south coast plain of California (Schlinger et al. 1960, van den Bosch et al. 1962, van den Bosch et al. 1970). In 1969, a new heat tolerant biotype of I. pallidus was introduced from Iran and this strain rapidly established and spread throughout the walnut growing regions of California, including the Central Valley (Frazer & van den Bosch 1973, van den Bosch et a!. 1979, Nowierski 1979). Recently, a new biotype (or possibly a sibling species) of I. Dallidus was introduced into Oregon from France to control filbert aphid; this new filbert aphid biotype will interbreed in the laboratory with I. pallid us from the walnut aphid (Messing & Alinlazee 1988). Variability in responses to azinphosmethyl (Guthion) in I. pallldus populations collected from walnuts in the Central Valley of California has been documented (Hoy & Cave 1988). Thus, it appears that the I. pallidus parasitoid group is genetically complex and could include sibling/cryptic species. At the least, populations may vary in host preferences, temperature tolerances, and tolerances to pesticides. It is difficult to integrate biological and chemical control in integrated pest management (IPM) programs. The incompatibility of these two types of control often results in pest resurgences and secondary pest outbreaks. The use of ecologically or physiologically selective insecticides to preserve natural enemies can overcome some of these incompatibilities (Mullin & Croft 1985, Hull & Beers 1985, Horn & Wadleigh 1988). In other cases, the use of natural or artificially-selected pesticide-resistant strains of natural enemies can enhance the compatibility of chemical and biological controls (Hoy 1979, 1982, Beckendorf & Hoy 1985). In California walnut orchards, the codling moth, ~ Domonella (L.) or navel orangeworm, Amvelois transitella Walker, can be controlled with azinphosmethyl (Guthlon), but azinphosmethyl has often been associated with secondary outbreaks of the walnut aphid (Riedl et al. 1979, Sibbett et a!. 1981). During 1985 and 1986, we collected colonies of I. pallidus from walnut orchards in California and determined that variability in responses to azinphosmethyl existed in these wild populations of I. pallidus. although field usable levels of azinphosmethyl resistance were apparently absl;tnt (Hoy & Cave 1988). We subsequently

81 selectedan azinphosmethyl-resistantstrain in the laboratory (Hoy & Cave 1988) and released it into five commercial walnut blocks during 1988 (Hoy et aI., in preparation). This paper reports the toxicity of five pesticides used in walnut IPM to a wild (Base) colony and the laboratory-selected azinphosmethyl-resistant (Selected) strain of I. pallid us. Tests were conducted by placing adult parasites in a clip cage on field-treated walnut foliage; survival on field rates was determined after 4, 8, 18, 24, 48, and 72 hrs on residues one to 35 days old. Materials and Methods Colony Sources and Culture Methods. The Selected colony of I. pallidus was selected in the laboratory for resistance to azinphosmethyl (Hoy & Cave 1988, in preparation). The Base colony was derived by pooling unselected field-collected colonies from commercial California walnut orchards (Hoy & Cave 1988). Both colonies of I. pallidus were maintained on walnut aphids reared on potted seedling Persian walnut trees grown in a University of California greenhouse at Berkeley. Synchronous I. pallidus colonies were reared on the potted trees containing walnut aphids in cloth-covered sleeve cages (45 X 45 X 86 cm) at 27 :I:.1.50C under continuous light. Assay Methods. Parasites were exposed to pesticide residues by holding them in clip cages attached to field-collected walnut leaves. Clip cages were made from 31.5 mm (inner diameter) clear acrylic tubing cut into 8 mm rings. One side of the ring was padded with 12 mm thick foam to cushion and provide a seal against the leaf surface. The other side of the ring was covered with cloth mesh to allow air circulation. The cage was attached to the underside of the walnut leaf with a hair clip (Hoy & Cave 1988). Parasites were anesthesized with carbon dioxide for approximately 1-3 minutes so they could be placed into the cages. Ten parasites (both sexes) were placed in each cage and 100 parasites per colony were tested, except that only 50 Base parasites were tested on the 15-day-old residues collected from the Hanford orchard. One hundred parasites were tested on untreated walnut foliage as controls (except only 15 were treated on the 15-day-old residues collected from the Hanford orchard). Survival was scored after 4, 8, 18, 24, 48, and 72 h exposure to the residues. Cages were held at 27 :I:. 1.50C under continuous light. Pesticides and Rates Tested. Two series of tests were conducted: foliage was collected from two commercial walnut orchards where phosalone, methidathion, or azinphosmethyl were applied by the grower using commercial spray equipment (Table 1). Trees sampled in the Colusa orchard were Serr, Chico, Vina, and Tehama varieties. Trees sampled in the Hanford orchard were Vina and Chico varieties. The second series of tests were conducted in an experimental Hartley variety walnut block near Davis, California. Field rates of azinphosmethyl, chlorpyrifos, endosulfan, methidathion, and phosalone were applied to drip by a single applicator to individual trees using a hand gun (Table 1). One or two untreated trees served as a buffer between treatments. This test allowed simultaneous comparison of residues of azinphosmethyl, chlorpyrifos, endosulfan, methidathion, and phosalone after 1, 14, and 28 days, in those cases where the pesticides remained toxic after 14 days. Survival of the Selected and Base colonies on each pesticide residue were compared using the Mantel and Haenszel's Chi-Square test (Lee 1980). Results Toxicity of Treated Foliage from the Experimental Block. The azinphosmethyl- resistant (Selected) and wild (Base) colonies of I. pallid us differed in their responses to all pesticides. The Selected colony consistently survived better on all residues, except the 14- day-old endosulfan residue, indicating that selection for azinphosmethyl resistance resulted in cross resistances to chlorpyrifos, endosulfan, methidathion, and phosalone (Table 2). On fresh (one-day-old) residues the Selected colony exhibited 22, 3, 92, 43, and 76% survival rates, respectively, after spending 72 h on azinphosmethyl, chlorpyrifos, endosulfan, methidathion, and phosalone residues. In contrast, the Base colony exhibited 1, 0, 34, 4, and 3% survival rates after spending 72 h on these residues. Chlorpyrifos residues were so toxic to the Base

82 colony that after 4 h, only 1% survived, but 36% of the Selected parasites were alive after the same interval (Table 2). The Selected and Base colonies exhibited significantly different survival rates on chlorpyrifos, azinphosmethyl, and methidathion residues that were 14 days old (Table 2). The Selected colony exhibited a high survival rate (90 and 94%) on chlorpyrifos and methidathion, respectively, compared to only 10 and 23% for the Base colony after 72 h. The high survival rate on both chlorpyrifos and methidathion by the Selected colony suggests that these two pesticides would select for the Selected colony in walnut orchards, even after 14 days. The survival of the Base colony on 14-day-old residues of endosulfan and phosalone suggests that these products are relatively more selective to the wild populations of I. pallidus because residues persist a shorter time (Table 2). Selected and Base colonies exhibited different survival rates on azinphosmethyl and methidathion residues that were 28 days old (Table 2). Survival of the Selected colony on 28- day-old azinphosmethyl residues after 72 h was 36% compared to 7% for the Base colony. Azinphosmethyl clearly has a long residual action. Survival on methidathion after 72 h was 97 and 56%, respectively, for the Selected and Base colonies, suggesting that methidathion may select for the Selected colony in walnut orchards even after 28 days. Chlorpyrifos residues 28 days old allowed 98 and 81% survival rates, respectively, after the Selected and Base parasites were on the residues 72 h, which suggests that this pesticide has a shorter residual than azinphosmethyl or methidathion. Pesticide residues remain toxic to the wild (Base) colony of I. pallidus for different intervals, with azinphosmethyl the most toxic for the longest interval and methidathion, chlorpyrifos, phosalone, and endosulfan less toxic in that order (Table 2). For the Selected colony, azinphosmethyl is also the most toxic for the longest time, but 22, 49, and 36% of the Selected parasites survived 72 h on 1-, 14-, and 28-day-old residues, respectively. Phosalone and endosulfan were nearly nontoxic to the Selected strain; 91 and 95% survived on 1- and 14-day-old residues after 72 h. For phosalone, 76 and 100% of the parasites survived 72 h on 1- and 14-day-old residues, respectively. Survival of the Selected strain on methidathion residues was nearly as good, with 43 and 94% surviving 72 h on 1- and 14-day-old residues, respectively. Chlorpyrifos residues declined sharply in their toxicity to the Selected strain between 1 and 14 days; survival increased from 3% on 1-day-old residues to 90% on the 14- day-old residues (Table 2).

Results of Foliage Tests from Commercial Walnut Orchards. The results obtained by sampling foliage treated with azinphosmethyl, methidathion, and phosalone from commercial walnut orchards generally confirm the results obtained above despite the fact that different concentrations of pesticides were applied with different equipment (Tables 1, 3). The Selected and Base colonies responded differently to residues of azinphosmethyl, methidathion, and phosalone (Table 3), with the Selected colony surviving better in all cases. Again, azinphosmethyl was the most toxic pesticide tested, with methidathion and phosalone less toxic in that order. Azinphosmethyl residues from the Hanford orchard remained highly toxic to the Base colony of I. Dallidus 35 days after application (Table 3). After 24 h, none of the Base parasites survived, but 41% of the Selected colony remained alive. Survival of the Selected colony on the azinphosmethyl residues declined to 2% after 72 h. However, the Selected colony consistently survived better than the Base strain on all azinphosmethyl residues (5, 15, or 35 days old). It is interesting to note that the residues that were 35 days old were essentially as toxic as the residues that were five days old. The reason for the higher survival rates of both colonies on the 15-day-old residues is unknown, but could be due to sampling foliage with less residue or differences in vigor of the parasites tested, or other unknown experimental variation. Survival of the Base colony of I. pallidus after 72 h on methidathion-treated foliage from the three walnut blocks was 0% (Table 3). Survival of the Selected strain after 72 h was

83 more variable, ranging from 5 to 47% in the three blocks. Residues 15 days old from the Hanford orchard allowed 88% survival of the Selected strain, but only 36% survival of the Base colony (Table 3). This is close to the survival of the two colonies on the 14-day-old residues from the experimental block (Table 2, 94 and 23%). Five- and 15-day-old residues of phosalone (Zolone) from the Hanford orchard were more toxic to the Base than the Selected colony of I. pallidus (Table 3). The survival of the Base colony in the Hanford block (Table 3) is lower than the survival in the experimental block (Table 2), but the differences could be explained by the greater concentration applied in the Hanford block than in the experimental block, differences in spray equipment and coverage, or differences in walnut variety foliage (Table 1). Discussion Selection for azinphosmethyl resistance in I. pallidus apparently has yielded cross resistances to chlorpyrifos, endosulfan, methidathion, and phosalone because the Selected strain consistently survives better on these pesticide residues than the wild (Base) colony. The mode of inheritance and mechanism(s) of the azinphosmethyl resistance are unknown at this time, making a discussion of the biochemical basis for the cross resistances premature. These cross resistances should facilitate implementation of the azinphosmethyl- resistant strain of I. pallidus in walnut orchards. Persistence of the genetically-manipulated azinphosmethyl-resistant strain of I. pallidus in an orchard is dependent upon a combination of factors, including the fitness of the resistant strain, the mode of inheritance and stability of the azinphosmethyl resistance, the degree of interbreeding with surrounding susceptible populations, and the selection intensity in favor of the resistant strain. If azinphosmethyl is applied to control second brood codling moth (usually in June), the long-lasting residues should favor the retention of the azinphosmethyl-resistant strain and continue to cause mortality to the wild strain (Tables 2, 3). If growers apply methidathion to control scale insects during the growing season, this should also favor the retention of the azinphosmethyl-resistant strain since there is a substantial differential in survival of the Selected and wild colonies for at least two weeks. Even applications of phosalone or endosulfan, which are considered to be less disruptive to wild I. pallidus, may favor the Selected strain, because these materials are also more toxic to the wild than the Selected strain for approximately two weeks. The data suggest that establishment and implementation of the Selected strain may not require an application of azinphosmethyl. Since all of the pesticides tested are commonly applied in walnut IPM, application of one or more of them during the growing season could aid the establishment and persistance of the azinphosmethyl-resistant (Selected) strain of I. pallidus in California walnut orchards. The data obtained with the wild strain of I. pallidus have implications for walnut IPM. The lengthy negative effects of azinphosmethyl on the wild (Base) strain explains, at least in part, the often-observed secondary outbreaks of walnut aphids where azinphosmethyl is applied. Phosalone (Zolone) is recommended for use against codling moth in walnut orchards because it is considered to be less disruptive to I. pallidus than azinphosmethyl (Sibbett et al. 1981). It is also considered to be a useful aphidicide. These data indicate that fresh residues of phosalone are more toxic to the wild (Base) colony of I.. pallidus than they are to the laboratory-selected azinphosmethyl-resistant strain. Phosalone residues are not highly toxic after about 14 days to the Selected colony. Survival after 72 h on 14-day-old residues from the experimental block was 86% (Table 2) and 12% after 72 h on 15-day-old residues in the Hanford block (Table 3). This difference is unexplained but suggests that phosalone could remain toxic to the wild strain of I. pallidus for two weeks. Perhaps the relatively short residual activity of phosalone to I. pallid us, in conjunction with its aphidicidal properties, is responsible for the perceived selectivity of phosalone to the wild colonies of I. pallid us. Methidathion (Supracide) is periodically applied to control scale insects in walnut orchards, and is considered to be disruptive to L pallidus. Survival of the Base colony after 72

84 --- - h on 28-day-old methidathion residues was 56% (Table 2), suggesting that methidathion could be applied in ways to enhance selectivity to I. pallidus. If methidathion were applied in strips or alternate rows in walnut orchards, wild I. pallidus populations might be preserved in the untreated reservoirs. I. pallidus could then recolonize the treated portion of the orchard within a month or so from the untreated reservoirs. Endosulfan (Thiodan) has a relatively short residual activity against the wild strain of I. pallidus (Table 2). This material is applied to control aphids, and its use has rarely resulted in aphid resurgences. Perhaps this is due, in part, to the fact that the residues are not highly toxic to I. pallid us, thereby allowing I. pallid us to control residual patches of aphids not controlled by endosulfan. Chlorpyrifos (Lorsban) is applied to control codling moth and navel orangeworm and the residues are highly toxic to the wild strain of I. pallid us. After 172 h the wild strain has only a 10% survival rate on 14-day-old residues (Tabl~ 2). Based on these data, we would expect chlorpyrifos, like azinphosmethyl, to stimulate aphid outbreaks in walnut orchards through destruction of the parasite populations, although, because chlorpyrifos is less toxic after 28 days than azinphosmethyl to the Base colony (Table 2), we would predict that it could have less negative impact than azinphosmethyl. These data describe the effects of different aged residues of pesticides used in walnut IPM to I. pallid us adults using a clip cage on field-treated walnut foliage. While this bioassay technique is an improvement in assessing direct mortality over an earlier method using a plastic cup (Hoy & Cave 1988), the relationship between these results and mortality under field conditions is unknown because biotic and abiotic factors can vary substantially from laboratory test conditions. The test also provides no information on the effect of the pesticides on aphids, or sublethal or indirect effects on I. pallidus. As noted by Messing et al (1988), such indirect effects can have significant impact on host and parasitoid populations. However, the information obtained may allow growers and pest control advisors to begin to make more informed decisions about using pesticides commonly used in walnut IPM. The data can also serve as a basis for additional studies designed to enhance the biological control of walnut aphid in California walnut orchards with the genetically-selected azinphosmethyl-resistant strain of I. pallidus. Acknowledgment We thank K. Spollen, R. Beede, W. Olson, W. Kreuger, and B. Bisabri for assistance in obtaining field-treated foliage for testing,T. Speed, Department of Statistics, University of Calfiornia, Berkeley for statistical advice, and E. Brown for assistance with the tests. This project was supported in part by the California Agricultural Experiment Station, Walnut Marketing Board, and Western Regional Research Project W-84.

References Cited Beckendorf, S. K. & M. A. Hoy. 1985. Genetic improvement of arthropod natural enemies through selection, hybridization or genetic engineering techniques, 167-187, In. M. A. Hoy & D. C. Herzog, eds., Biological Control in Agricultural IPM Systems, Academic Press, N.Y.

Frazer, B. D. & R. van den Bosch. 1973. Biological control of the walnut aphid in California; the interelationship of the aphid and its parasite. Environ. Entomol. 2: 561-568.

Horn, D.J. & R. W. Wadleigh. 1988. Resistance of aphid natural enemies to insecticides. p. 337- 347. 1n A. K. Minks & P. Harrewijn, eds., Aphids, Their Biology, Natural Enemies and Control, Vol. B, Elsevier, Amsterdam.

Hoy, M. A. 1979. The potential for genetic improvement of predators for pest management programs, 106-115. 1n M. A. Hoy & J. J. McKelvey, Jr., eds., Genetics in Relation to Insect Management. Rockefeller Foundation Press, N. Y.

85 Hoy, M. A. 1982. Genetics and genetic improvement of the Phytoseiidae, 72-89. in M. Hoy, ed., Recent Advances in Knowledge of the Phytoseiidae, Publ. 3284, Univ. of Calif. Div. Agric. Sci, Berkeley, CA.

Hoy, M. A. Pesticide resistance in arthropod natural enemies: variability and selection responses. In R. T. Roush & B. Tabashnik, eds., Pesticide Resistance in Arthropods, in press.

Hoy, M. A. & F. E. Cave. 1988. Guthion-resistant strain of walnut aphid parasite. California Agriculture 42 (4): 4-5. .

Hull, L. A. & E. H. Beers. 1985. Ecological selectivity: modifying chemical control practices to preserve natural enemies, 103-122, 1n.M. A. Hoy & D. C. Herzog, eds., Biological Control in Agricultural IPM Systems, Academic Press, N. Y.

Lee, E.T. 1980. Statistical Methods for Survival Data Analysis, Wadsworth.

Messing, R. H. & M. T. Aliniazee. 1988. Hybridization and host suitability of two biotypes of Trioxvs pallidus (Hymenoptera: Aphidiidae). Ann. Entomol. Soc. Am. 81: 6-9.

Messing, R. H., M. T. AIiNiazee & J. Calkin. 1988. Indirect effects of carbaryl on populations of the filbert aphid, Mvzocallis ~ (Goeze) (Hom., Aphididae). J. Appl. Ent. 106: 72-78.

Michelbacher, A. E. & J. C. Ortega. 1958. A technical study of insects and related pests attacking walnuts. Univ. Calif. Bull. 764:1-87.

Mullin, C. A. & B. A. Croft. 1985. An update on development of selective pesticides favoring arthropod natural enemies, 123-150, 1n.M. A. Hoy & D. C. Herzog, eds., Biological Control in Agricultural IPM Systems, Academic Press, N. Y.

Nowierski, R. M. 1979. The field ecology of the walnut aphid, Chromaphis juolandicola (Homoptera: Aphididae) and its introduced parasite, Trioxys pallidus (Hymenoptera: Aphidiidae)--a qualitative and quantitative assessment of population regulation. Ph.D. thesis, University of California, Berkeley, 231 pp.

Riedl, H., M. M. Barnes & C. S. Davis. 1979. Walnut pest management: historical perspective and present status, pp. 15-80. 1n.D. M. Boethel & R. D. Eikenbary, eds., Pest Management Programs for Deciduous Tree Fruits and Nuts, Plenum Press, N. Y.

Schlinger, E. I., K. S. Hagen & R. van den Bosch. 1960. Imported French parasite of walnut aphid established in California. California Agriculture 14(11): 3-4.

Sibbett, G. S., L. Bettiga & M. Bailey. 1981. Impact of summer infestation of walnut aphid on quality. Sun-Diamond Grower, June-July, 8-9, 50.

UC/IPM. 1982. Integrated Pest Management for Walnuts, Univ. Calif. Div. Agric. Sci. Publ. 3270, Berkeley, CA.

van den Bosch, R., E. I. Schlinger & K. S. Hagen. 1962. Initial field observations in California on Trioxvs callidus (Haliday) a recently introduced parasite of the walnut aphid. J. Econ. Entomol. 55: 857-62.

86 --- - - van den Bosch, R., B. D. Frazer, C. S. Davis, P. S. Messenger & R. Horn. 1970. Trioxys pallidus an effective walnut aphid parasite from Iran. California Agriculture 24: 8.10. van den Bosch, R., R. Horn, P. Matteson, B. D. Frazer, P. S. Messenger & C. S. Davis. 979. Biologicalcontrol of the walnut aphid in California:impact of the parasite, Trioxvs S2.a1~. Hilgardia 47(1): 1.13.

Table 1. Pesticide rates and application methods in the commercial and experimental Persian walnut orchards in California

Orchard location Pest i ci de Rate and application method and variety (formulation)

1) Experimental orchard!/ Davis azinphosmethyl 1.0 lb AI/400 gal/Acre (Guthion 50 WP) handgun Hartley (1.12 kg AI/3741 l/hectare) chlorpyrifos 2.0 lb AI/400 gal/Acre (Lorsbon 4E) handgun (2.24 kg AI/3741 l/hectare) endosuIf an 1.5 lb AI/400 gal/Acre (Thiodan 50WP) handgun (1.68 kg AI/3741 l/hectare) methidathion 2.0 lb AI/400 gal/Acre (Supraci de 2E) handgun (2.24 kg AI/3741 l/hectare) phosalone 2.25 lb AI/400 gal/Acre (Zolone 3E) handgun (2.52 kg AI/3741 l/hectare)

2) Commercial orchards

Hanford azinphosmethyl 1.5 lb AI/250 gal/Acre Vina & Chico (Guthion 50WP) (1.68 kg AI/2338 l/hectare) Hanford methidathion 2.0 lb AI/250 gal/Acre Vina & Chico (Supracide 2E) (2.24 kg AI/2338 l/hectare) Hanford phosalone 2.25 lb AI/250 gal/Acre Vina & Chico (Zolone 3E) (2.52 kg AI/2338 l/hectare) Colusa North block methidathion 1.5 lb AI/120 gal/Acre Serr, Chico, Vina (Supracide 2E) windmill sprayer & Tehama (1.68 kg AI/1122 l/hectare) Colusa South block methidathion 1.5 lb AI/120 gal/Acre Serr, Chico, Vina (Supracide 2E) windmill sprayer & Tehama (1.68 kg AI/1122 l/hectare)

!/ Trees were sprayed to drip using ca. 7.9 gal/tree.

- --- Table 2. Survival of the wild (Base) and azinphosmethyl-resistant (Selected) colonies of I. pallidus on 1-, 14- and 28-day old pesticide residues from an experimental walnut orchard near Davis, California. Tests were conducted at 27° t 1.5°C under continuous light with clip cages; survival was assessed after 4, 8, 18, 24, 48 and 72 h

Pesticide!1 Survival % survival on residues aqed1 scored after (h) 1 14 28 days

Selected Base Selected Base Selected Base

azinphosmethyl 4 90 54 100 86 96 91 8 86 33 99 72 94 78 18 58 14 93 38 88 41 24 48 9 85 30 83 24 48 28 2 59 16 59 9 72 22 1 49 10 36 7

chlorpyrifos 4 36 1 99 85 100 99 8 18 0 99 67 100 99 18 12 0 98 52 99 98 24 9 0 96 40 99 98 48 4 0 95 18 99 92 72 3 0 90 10 98 81

endosulfan 4 100 73 99 97 8 98 55 99 96 18 97 52 98 96 24 94 47 98 96 48 92 36 97 96 72 91 34 95 94

methidathion 4 90 57 100 93 99 99 8 82 45 99 81 98 99 18 69 21 99 64 98 91 24 61 14 99 54 98 84 48 48 10 98 36 97 66 72 43 4 94 23 97 56

phosalone 4 99 83 100 100 8 98 56 100 100 18 96 31 100 99 24 95 27 100 97 48 89 18 100 92 72 76 3 100 86

!I Pesticide rates and application methods are given in Table 1.

I Each test was conducted using 10 I. pallidus in each of 10 cages. 100 parasites were tested on untreated foliage as controls; control mortality was never greater than 5% so is not reported. Survival of the Selected and Base colonies was compared for each pesticide residue age by the Mantel and Haenszel's chi-square test (Lee 1980). The Selected colony survived better than the Base colony (P < 0.05) on all residues except for the 14-day-old endosulfan residues for which there was no significant difference.

------Table 3. Survival of the wild (Base) and azinphosmethyl-resistant (Selected) strains of T. pallidus on different aged residues of pesticide-treated walnut foliage from commercial - orchards. Tests were conducted at 27°C t 1.5°C under continuous light with clip cages and survival was assessed after 4, B, 18, 24, 48 and 72 h

Orchard Pesticlde!1 Survival % survival on residues aqed21 scored after (h) 5 15 35 days

Selected Base Selected Base Selected Base Colonl

Hanford azinphosmethyl 4 97 71 99 54 96 71 8 93 42 95 37 87 31 18 73 10 88 12 71 8 24 57 4 83 8 41 0 48 21 1 44 6 5 0 72 ." 1 24 6 2 0

Hanford methidathlon 4 86 10 99 100 8 47 4 96 90 18 20 0 94 66 24 11 0 92 60 48 6 0 90 44 72 5 0 88 36

Hanford phosalone 4 99 97 98 86 8 99 __ 86 95 78 18 99 58 95 63 24 99 37 92 57 48 97 16 88 16 72 94 11 85 12

Colusa methldathlon

North block. 4 92 86 8 88 64 18 84 26 24 78 6 48 57 2 72 47 0

South block. 4 96 12 8 86 5 18 54 2 24 43 2 48 16 0 72 15 0

!I Pesticide rates and application methods are given in Table 1.

2/ Each test was conducted using 10 !. pallidus In each of 10 cages. 100 parasites were tested on untreated foliage as controls; control mortality was never greater than 5% so is not reported. Survival of .the Selected and Base colonies was compared for each pesticide residue age by the Mantel and Haenszel's chi-square test (lee 1980); the Selected colony survived better than the Base colony on all ages of residues (P < 0.05).

-- --- Release, Dispersal, and Recovery of a Laboratory-Selected Azlnphosmethyl-Reslstant Strain of the Walnut Aphid Parasite Trloxvs paliidus (Hymenoptera: Aphldlldae)

M. A. Hoy,1 F. E. Cave,1 R. Beede,2 J. Grant,3 W. Krueger,4 W. 0lson,5 K. Spollen,1 W. W. Barnett,6and L. C. Hendricks7

ABSTRACT A strain of Trioxvs pallidus, selected in the laboratory for resistance to azinphosmethyl, was mass reared and released into five commercial walnut blocks in California to determine whether the strain could survive field applications of azinphosmethyl or methidathion, persist, and parasitize the walnut aphid, Chromaphis jUQlandicola. Approximately 75,000 parasites were reared on potted walnut trees in a University of California greenhouse in Berkeley; releases were made after azinphosmethyl applications in three sites, after methidathion application in one site, and just before methidathion application in one site. Colonies of I. pallidus collected from each orchard prior to the releases were tested with a discriminating dose of azinphosmethyl to determine resistance levels of the wild population. Samples of I. pallidus were collected once or twice from each block after releases were made, and from adjacent walnut blocks in two sites, and assayed with azinphosmethyl. The high survival of the parasites indicated the azinphosmethyl-resistant strain of 1. pallidus established and persisted through the growing season in four of the five walnut blocks and dispersed to adjacent nonrelease blocks in two of two sites sampled. Counts of aphids and mummies support the conclusion that the laboratory-selected strain survived on field rates of azinphosmethyl or methidathion, parasitized aphids, and persisted within the release and nearby nonrelease sites during the 1988 growingseason.

Kev words: genetic improvement, azinphosmethyl, walnut aphid, resistance, Trioxvs pallidus

GENETIC improvement of arthropod natural enemies has been discussed extensively, but relatively few laboratory-selected natural enemies have been field tested (Beckendorf & Hoy 1985, Hoy 1986). Genetic manipulation will remain a controversial tactic in biological control until we can quantify the likelihoodof achieving successful selections, and document the fitness and efficacy of genetically-selected natural enemies under field conditions. Genetic manipulation of parasitic Hymenoptera is thought to provide a particularly difficult challenge because parasites are perceived to have low levels of genetic variability (Grauer 1985) and are thus unlikely to respond to selection. An additional concern is the low probability of achieving field-usable levels of resistance to pesticides in parasites, as they have rarely been documented to develop resistance (Croft & Strickler 1983, Horn & Wadleigh 1988). Trioxvs pallidus Haliday is a highly effective parasite of the walnut aphid, ChromaDhis jUQlandicola(Kaltenbach), in Californiawalnut orchards. Two biotypes of I. pallidus have been released; one was obtained from France and the other from Iran (Schlinger et al. 1960, van den Bosch et al. 1962, 1970, 1979). The Iranian strain is generally credited with being more tolerantof hot dry conditionsthan the Frenchstrain and this tolerance is considered key to its rapid establishment and impact on the walnut aphids in 1969-1970 in California's hot, dry Central Valley. Unfortunately, I. pallidus is susceptible to azinphosmethyl, and use of this product to control codling moth, ~ pomonella L., or navel orangeworm, Amevlois transitella Walker, can result in disruption of I. pallidus and lead to secondary outbreaks of walnut aphids (Michelbacher & Ortega 1958, Riedl et al. 1979, Sibbett et al. 1981). We thus began a genetic selection project with I. pallidus to determine whether we could develop an

- azinphosmethyl-resistant strain. In 1985 and 1986, colonies of I. pallidus were collected from California walnut orchards and screened to determine if variability in responses to azinphosmethyl was present (Hoy & Cave 1988). Substantial variability was found, laboratory selection was successful, and the resistance level achieved appeared to be sufficiently high that field trials were warranted (Hoy & Cave 1988). We report the results of releasing the laboratory-selected strain of azinphosmethyl- resistant I. pallidus into five commercial walnut blocks in California to determine whether this strain could survive on pesticide-treated foliage, parasitize walnut aphids, and persist during the growing season. Parasites were sampled from nearby walnut blocks at two sites to determine whether the resistant strain had dispersed from the release sites. Materials and Methods Colony Sources and Culture Methods. The Selected colony of I. pallidus was selected in the laboratory for resistance to azinphosmethyl (Hoy & Cave 1988). The Base colony was derived by pooling field-collected colonies from commercial California walnut orchards (Hoy & Cave 1988). Both colonies of I. pallidus were maintained on walnut aphids reared on potted seedling Persian walnut trees, JUQlanswla L., grown in a University of California greenhouse at Berkeley. Synchronous I. pallidus colonies were reared on the potted trees containing walnut aphids in cloth-covered sleeve cages (45 X 45 X 86 cm) at 27 .t. 1.50C under continuous light. Mass Rearing Methods. Approximately 75,000 azinphosmethyl-resistant I. pallidus were reared for releases made June 23-27, 1988. Franquette variety walnut seeds were planted in flats, twenty per flat, in late February and early March 1988 and grown under continuous light in the greenhouse until April, when they were transferred to 15.2 cm diameter pots and held under continuous light in a greenhouse cubicle. Seedling trees were monitored weekly and maintained free of spider mites by releasing the phytoseiid Metaseiulus occidentalis (Nesbitt). Walnut aphids were removed by hand. Trees were used after they had produced a terminal bud and five to seven leaves. On June 8-13, trees were infested with all stages of aphids. On June 14-16, 24 trees each day were moved to large cages (91.4 X 91.4 X 91.4 cm) in the laboratory (8 trees/cage) where approximately 450 to 500 1. pallid us (both sexes) were released into the cage for 24 h. The parasites were then removed by an aspirator using a vacuum pump. Trees containing the parasitized aphids were held in a cubicle in the greenhouse until mummies began to form. The number of parasites produced was estimated by counting mummies on the top and bottom sides of half of all the leaves of trees just prior to releases; mummies from all trees (15) being placed in the Stockton and Gustine sites, mummies on 8 of 24 trees being placed in the Hanford site, and on 4 of 33 trees going to the Colusa North and South blocks were counted. Releases were made by placing potted trees in two orchards (Hanford and Stockton), or by stapling foliage to tree trunks at three sites (Colusa North, Colusa South, and Gustine) and allowing the parasites to emerge. Releases were timed so that adult parasites should have emerged within 1-3 days after releases. Foliage from the Stockton release site was retrieved and the number of successful emergences estimated by counting the number of mummies with emergence holes and the numb~r of mummies with no holes. Release Sites. Releases were made into five commercial walnut blocks in the Central Valley of California (Table 1). Block size, tree varieties, cultural practices, and pesticide applications varied at each site (Table 1). Azinphosmethyl applications were made prior to releases at the Hanford, Stockton, and Gustine sites. Methidathion was applied to the Colusa North block prior to parasite releases and after parasite releases into the Colusa South block. The azinphosmethyl-resistant strain of I. pallidus is cross resistant to methidathion (Hoy & Cave, submitted). Parasite Sampling Methods. I. pallid us was collected from the release sites before and after the azinphosmethyl-resistant strain was released; this allowed us to determine whether the native population was tolerant of azinphosmet~yl and whether the released population was present (Figs. 1-5, Table 2). Foliage with aphids and/or mummies was collected, placed in an ice chest, chilled, and returned to the laboratory. Mummies were removed with forceps from

91 - --- the foliage and placed in a one-ounce plastic cup with honey and held until parasites emerged; any hyperparasites were then eliminated. In addition, leaves with aphids were cut, placed on water-soaked cotton in plastic dishes and held at 270C until mummies formed; these mummies were rarely hyperparasitized. After adults emerged, the parasites were counted and sexed, and used to initiate a new colony. Colonies were reared on potted walnut trees infested with walnut aphids and held in cages (45 X 45 X 86 em) at 270C under continuous light. Progeny were tested to determine their tolerance of azinphosmethyl as described below. Plastic Cup Bioassay. Azinphosmethyl (Guthion 50WP) was dissolved in 95% (190 proof) ethanol to form a concentrated stock solution. Stock solutions were made fresh each test day. A dilute solution of 50 ppm A.I. was made from the stock. One-ounce plastic cups were treated by filling them with the dilute pesticide solution for 5 seconds, pouring the solution out, and inverting the cup on paper toweling to dry. Control cups were treated with 95% ethanol. Two pieces of black vinyl electrical tape ca. 1.5 X 12 mm were placed on the inside of each treated cup and streaked with two or three lines of undiluted honey. Adult parasites that had emerged within 0-48 h were aspirated into the test cups. An untreated mesh cover for each cup was held on with a plastic cap which had all but the rim removed. Suction for the aspirator was provided by a vacuum pump that could be adjusted to minimize injury to the parasites. To minimize contamination, the rubber stopper of the aspirator was covered with organdy cloth and parasites were added to the vials starting with the water control and then the test concentration. The cloth was discarded and rubber stopper cleaned after each test. Tests were conducted with 10 (or 20) parasites (both sexes) per cup and survival was assayed after 24 h at 250C under 16L:8D cycle. Aphid and Mummy Counts. Mature foliage was monitored every week to two weeks to estimate the numbers of aphids and mummies present in the release and nonrelease sites. Ten leaves were sampled from each marked sample tree; leaves were cut with a pole pruner from around the tree up to approximately five meters. The number of aphids and mummies on one penultimate leaflet (of a leaf sampled from just behind a walnut) were counted and recorded (UC/IPM 1982). Ten marked trees were sampled from Release and adjacent Nonrelease sites in the Stockton and Gustine sites, ten trees only in the Colusa North and Colusa South blocks, and six trees where releases were made in the Release block, eight Sample trees in the Release block, and eight trees in the nearby Nonrelease block at the Hanford site. Results Rearing. Approximately 75,000 azinphosmethyl-resistant I. pallidus were reared on a total of 72 potted trees for release into the five blocks (Table 1). The potted trees produced 350 to 1900 parasites per tree. Approximately 9000 I. pallidus mummies were released into the 3-acre (1.2 hectare) Stockton site on June 23, 1800 into the 2-acre (0.8 hectare) Gustine site on June 23, 30,000 into the 2.5-acre (1.01 hectare) Hanford site on June 24, and 12,000 into the 17-acre (6.88 hectare) Colusa North, and 24,000 into the 17-acre Colusa South blocks on June 27, 1988. Several problems were encountered in rearing the parasites. Beginning in April, a fungal disease (Entomopthora species) reduced walnut aphid numbers in the stock colony, and made dense infestation of the 72 walnut trees difficult. In addition, the aphid-infested trees were invaded by predatory cecidomyiids, which had to be removed by hand. Hot weather (to over 430C in the greenhouse cubicle) in June for one day overtaxed the greenhouse cooling system and caused an estimated 20% mortality of parasitized aphids. Several improvements in rearing methods will need to be made if mass production of this parasite is to be commercialized. Resistance levels of native T. pailidus. Colonies of I. pallidus were obtained from all release sites prior to releases of the azinphosmethyl-resistant strain. Survival of these pre- release colonies treated with 50 ppm azinphosmethyl ranged from 0 to 42% (Table 2). The colony from Gustine exhibited the highest survival rate with 42% of the parasites surviving 50 ppm azinphosmethyl, but this was still lower than the 94% survival of the the resistant (Selected) laboratory colony (Table 2). Survival of parasites collected from the Stockton site

-- was 0%, 2% from the Colusa blocks, and 16% from the Hanford site (Table 2). Survival of the Selected colony ranged from 77 to 94%, indicating clear differences existed between the wild parasites and the Selected colony. Release, Recovery, and Dispersal. The azinphosmethyl-resistant strain of 1.. pallidus established in four of the five walnut blocks, survived field rates of azinphosmethyl or methidathion, persisted through the growing season, and dispersed to adjacent nonrelease sites in two locations (Figs. 1-5, Table 2). Hanford block. Parasites were released on June 24 into six trees in the Hanford block; samples taken from these six Release trees (Fig. 1A) and from eight adjacent Sample trees (Fig. 1B) indicated that I. pallidus were present through the growing season in the Release block. I. pallidus mummies were also present in the nearby Nonrelease block throughout the growing season (Fig. 1C), despite the fact that azinphosmethyl was applied on June 8-9 to all trees in both the Release and Nonreleasearea (Table 1, Fig. 1). Samples taken from the Release and Nonrelease area prior to release of the azinphosmethyl-resistant strain indicated that the wild parasites were susceptible to azinphosmethyl(16% survival compared to 0 and 94% survival for the Base and Selected colonies, respectively, Table 2). Thus, we expected larger aphid populations might develop in the Nonrelease block than in the Release block due to the negative effects of azinphosmethyl on the wild I. pallidus. Surprisingly, aphid populations were not dramatically different (Fig. 1) and we attribute this to the fact that the azinphosmethyl- resistant strain rapidly established itself in the Nonrelease block. Parasites were collected from the Release and Nonrelease areas at Hanford on July 14 and October 11 and tested with azinphosmethyl. Survival was 81% and 80%, respectively, for parasites recovered from the Release and Nonrelease areas for the July 14 sample, and 58% and 69% for the Release and Nonrelease areas for the October 11 sample (Table 2). The high survival rates indicate that the azinphosmethyl-resistant strain had established and persisted in the Release area and had spread to the Nonrelease area by July 14. The decline in survival to 58% and 69% on the second (October 11) sample date could be due to intermixture with wild susceptible parasites or to sample errors.

Stockton. None of the wild parasites collected from the Stockton orchard on June 23 survived 50 ppm azinphosmethyl in the plastic cup assay (Table 2). In contrast, the Selected colony exhibited a 94% survival rate in this test (Table 2). About 9000 parasites were released on June 23 into this 3-acre block, and parasites were recovered from the Release block on July 7 and from both the Release and adjacent Nonrelease blocks on September 12 (Fig. 2). Parasites collected July 7 exhibited a 72% survival rate compared to 0 and 77% for the Base and Selected colonies, respectively, indicating the azinphosmethyl-resistant strain had successfully survived and reproduced in the orchard; survival of parasites from the Release and Nonrelease areas on September 12 were 46 and 35%, respectively; the pre-release colony, Base, and Selected control had 8, 7. and 90% survival rates, respectively (Table 2). Azinphosmethyl was applied twice to the Release and Nonrelease blocks (Table 1). and I. pallidus mummies were found throughout the growing season in both Release and Nonrelease trees (Fig. 2). Again, because the azinphosmethyl-resistant strain apparently spread to the Nonrelease block, dramatic differences in aphids did not occur in the Release and Nonrelease blocks. Mean aphids/leaflet peaked in early July at approximately 11.6 in the Release trees and 13.1 in the Nonrelease trees, which is below the treatment level of 15 aphids per leaflet (Figure 2). Later in August another population increase occurred with a mean of 7.4 and 16.6 aphids per leaflet in Release and Nonrelease blocks, respectively. These data indicate that the azinphosmethyl-resistant strain established in the Release area, persisted throughout the growing season despite two applications of azinphosmethyl, and spread to the adjacent Nonrelease area (Table 2, Figure 2A, B).

Colusa North Block. The azinphosmethyl-selected strain was able to establish and persist in a methidathion-treated walnut block near Colusa (Fig. 3). Approximately 12,000

-- azinphosmethyl-resistant parasites were released into the 17-acre Colusa North block on June 27 after a methidathion application was made on June 22 (Fig. 3). We were unable to recover any live wild parasites from this block prior to parasite releases, but did obtain parasites from the adjacent Colusa South block (Table 2), and these were susceptible to azinphosmethyl (2% survival). Methidathion-treated mummies collected from the North block failed to yield any living adults; this is consistent with the observation that fresh residues of methidathion are highly toxic to adults of wild colonies of I. pallid us (Hoy & Cave, submitted). The grower applied low rates of phosalone to alternate rows twice (August 2 and 18) despite the fact that fewer than 8 aphids/leaflet were present (Fig. 3). These applications reduced the numbers of aphids available to maintain the released parasites. However, samples taken on August 16 and October 12 indicated that the azinphosmethyl-resistant strain had established in the block (Fig. 3, Table 2). Survival of the recovered parasites averaged 61% and 52%, respectively, for the two sample dates compared to 5% and 11% for the pre-release colony (Table 2). Colusa South block. The azinphosmethyl-resistant strain was released into the Colusa South block on June 27 ~ methidathion was applied on July 13/14 . Because the native I. pallid us population was not reduced or eliminated in this block prior to releasing the azinphosmethyl-resistant strain, we had a low expectation that the resistant strain could establish in this block (Fig. 4). The number of unparasitized aphids available to the released strain was low at the time of release due to the large numbers of wild parasites present (Fig. 4). The colony collected from this block prior to the release was susceptible to azinphosmethyl (2% survival, Table 2). After the azinphosmethyl-resistant strain was released, parasite colonies were collected on August 16 and October 12; survival was 36% and 24%, compared to 5% and 11% for the pre-release colony (Table 2), indicating that the Selected strain was present in the block. Because wild I. pallidus were not eliminated or reduced prior to release of the azinphosmethyl-resistant strain, the data suggest that the released strain successfully competed for hosts for approximately two weeks prior to the methidathion application. It is also possible that azinphosmethyl-resistant parasites dispersed from the adjacent Colusa block after the methidathion was applied, but it is impossible to discriminate between these possibilities. In any case, the azinphosmethyl-resistant strain performed well under less than optimal release conditions. Gustlne block. The azinphosmethyl-resistant strain of I. pallid us apparently did not establish at the Gustine release site (Fig. 5, Table 2). Aphid numbers were very low throughout the growing season and this could have impeded establishment, as well as the fact that only 1800 parasites were released into the 2-acre site (Fig. 5). Parasites collected from this block prior to the release of the resistant strain exhibited a 42% survival rate when tested with 50 ppm azinphosmethyl, which is the highest of any colony collected from the field (Table 2). Survival of the parasite colony recovered on August 15 from the Release area was 26%. No parasites were recovered from the Nonrelease area.

Discussion The azinphosmethyl-resistant strain of I. pallidus established and persisted through the growing season in four of the five release sites (Figs. 1-4, Table 2). In addition, it dispersed to nearby Nonrelease sites in two orchards (Figs. 1, 2, Table 2). This suggests that the laboratory-selected strain is able to survive on foliage treated with field rates of azinphosmethyl or methidathion, parasitize aphids, and disperse. The resistant strain performed well in four geographically distant commercial walnut blocks that had different aphid densities and received different pesticide applications. Thus, the laboratory-selected strain fulfilled the goals set for it during the 1988 growing season. Its overwintering success during 1988-89 remains to be determined. Genetic improvement of arthropod natural enemies involves a series of steps (Hoy 1979, Beckendorf & Hoy 1985). First, the desired trait should be identified and variability must be found (or provided for) upon which selection can occur. It is highly desirable that the

------mode of inheritance and fitness of the putatively-improved strain be known prior to field release and evaluation. In the case of I. pallidus, we did not know the mode of inheritance of the azinphosmethyl resistance and life table comparisons of the resistant and base colonies have not yet been completed. However, no matter how fit (or unfit) the strain appears under laboratory conditions, the critical evaluation of fitness will be made under the field conditions in which the strain must ultimately function. To date the azinphosmethyl-resistant strain has achieved criteria which are essential if it is to be implemented in walnut IPM programs in California: survival on field rates of azinphosmethyl or methidathion, persistence in the orchard, and dispersal. Implementing the azinphosmethyl-resistant strain of I. pallidus into walnut IPM will probably require the long term establishment of the resistant strain in commercial walnut orchards. At present there are no commercial companies that produce and sell I. pallidus for release; mass rearing I. pallidus requires that we rear potted walnut trees and walnut aphid since both I. callidus and the walnut aphid are host specific. If I. pallid us were to be reared commercially, several improvements in rearing technology would be helpful. These include having selective pesticides to control spider mites and fungal diseases of the walnut aphid, as well as more effective temperature controls in the greenhouse. In addition, it would be helpful to have rearing facillities that permit the isolation of aphid-infested and uninfested walnut trees. The samples of I. pallid us obtained prior to release of the azinphosmethyl-resistant strain show that azinphosmethyl is more toxic to wild I. pallid us than to the laboratory- selected strain. The recovery of azinphosmethyl-resistant parasites from four of the five release blocks (and from two adjacent nonrelease blocks) suggests that the laboratory-selected strain is persisting on azinphosmethyl and methidathion residues and reproducing and dispersing from the release sites. Should the azinphosmethyl-resistant strain of I. pallidus overwinter, its dispersal, impact on aphid populations, maintenance of resistance levels, and persistance over several seasons will be evaluated. Long term implementation will require additional information, but the first steps have been taken to field test a genetically manipulated, pesticide-resistant parasite. Acknowledgments We thank M. Dettenhofer, E. Brown, and F. Perry for assistance with the project and G. Thomas for identification of the fungal disease. Partial support was provided by the California Agricultural Experiment Station, Regional Research Project W-84, and Walnut Marketing Board of California. We also thank walnut growers M. Podesta, W. Torrison, L. Bairstow, and (owner of Steele orchard) for providing our experimental walnut blocks. References Cited Beckendorf, S. K. & M. A. Hoy. 1985. Genetic improvement of arthropod natural enemies through selection, hybridization or genetic engineering techniques, pp. 167-187. In M. A. Hoy & D. C. Herzog, eds., Biological Control in Agricultural IPM Systems, Academic Press, N.Y. Croft, B. A. & K.A. Strickler. 1983. Natural enemy resistance to pesticides: documentation, characterization, theory and application. In Pest Resistance to Pesticides, G. P. Georghiou & T. Saito, eds., Plenum, N.Y. Grauer, D. 1985. Gene diversity in Hymenoptera. Evolution 39:190-199. Horn, D. J. & R. W. Wadleigh. 1988. Resistance of aphid natural enemies to insecticides. P. 337-347. In A. K. Minks & P. Harrewijn, eds., Aphids, Their Biology, Natural Enemies and Control, Vol. B., Elsevier, Amsterdam. Hoy, M. A. 1979. The potential for genetic improvement of predators for pest management programs, 106-115, In M. A. Hoy & J. J. McKelvey, Jr., eds., Genetics in Relation to Insect Management, Rockefeller Foundation Press, N.Y. Hoy, M. A. 1986. Use of genetic improvement in biological control. Agric. Ecosys. Environ. 15: 109-119.

-- - Hoy, M. A. & F. E. Cave. 1988. Guthion-resistant strain of walnut aphid parasite. California Agriculture 42 (4): 4-5. Hoy, M. A. & F. E. Cave. (submitted) Toxicity of pesticides used in walnuts to a wild and laboratory-selected azinphosmethyl-resistant strain of Trioxvs pallidus (Hymenoptera: Aphidiidae). J. Econ. Entomol. Michelbacher, A. E. & J. C. Ortega. 1958. A technical study of insects and related pests attacking walnuts. Univ. Calif. Bull. 764:1-87. Riedl, H., M. M. Barnes & C. S. Davis. 1979. Walnut pest management: historical perspective and present status. pp. 15-80. In. D. M. Boethel & R. D. Eikenbary, eds., Pest Management Programs for Deciduous Tree Fruits and Nuts, Plenum Press, N.Y. Schlinger, E. I., K. S. Hagen & R. van den Bosch. 1960. Imported French parasite of walnut aphid established in California. California Agriculture 14 (11): 3-4. Sibbett, G. S., L. Bettiga & M. Bailey. 1981. Impact of summer infestation of walnut aphid on quality. Sun-Diamond Grower, June-July, 8-9, 50. UCIIPM. 1982. Integrated Pest Management for Walnuts, Univ. Calif. Div. Agric. Sci. Publ. 3270, Berkeley, CA. van den Bosch, R., E. I. Schlinger & K. S. Hagen. 1962. Initial field observations in California on Trioxvs Dallidus (Haliday) a recently introduced parasite of the walnut aphid. J. Econ. Entomol. 55: 857-62. van den Bosch, R., B. D. Frazer, C. S. Davis, P. S. Messenger & R. Horn. 1970. Trioxvs pallidus an effective walnut aphid parasite from Iran. California Agriculture 24: 8-10. van den Bosch, R., R. Hom, P. Matteson, B. D. Frazer, P. S. Messenger & C. S. Davis. 1979. Biological control of the walnut aphid in California: impact of the parasite, Trioxvs pallidus. Hilgardia 47(1): 1-13.

96 A. Releasetrees 30-I . S 3.0 m-- aphids. .-- mummies\ 20 2.0

10 1.0

o 0.0

B. Sample trees 30 3.0 - en Q) 20 2.0 E E ::J E 10 1.0 c: ~ Q) E o 0.0

C. Non-release 30 trees 3.0

20 2.0 S 10 AS 1.0 \ \ o 0.0 4 18 2 1630132711 25 8 22 5 19 3 17 31 Apr May Jun Jul Aug Sap Oct

Sample Date (1988)

Figure 1. Mean walnut aphids and I. pallidus mummies per leaflet from Release and Sample trees in the Hanford Release (A, 8) site and adjacent Nome/ease site (C). A=azinphosmethyl propargiteapplication application., R= parasites released , $= parasites sampled for laboratory bioassays, 0 = 97 ------30 . .3 A. Release trees m- aphids .- mummies 201 SA r-2 - II A 10 1 - Q) -Q) ;;: ;;: I CI3 CI3 - - CJ) CJ) 0 0 . "C :c E Co 30 3 E CI3 B. Non-release trees :;] c: E CI3 c: Q) A CI3 E Q) 20 2 E I X S 101 A I I \ I \ I 1-1

- 0 0 4 18 2 16 30 13 27 11 25 8 22 5 19 3 17 Apr May Jun Jul Aug Sep Oct

Sample Date (1988)

Figure 2. Mean walnut aphids and I. pallidus mummies per leaflet from the Stockton Release (A) and Nonrelease (B) blocks. A= azinphosmethyl applications, S=parasites sampled for bioassays, R= azinphosmethyl-resistant parasites released.

------40 4 - aphids Q) -Q) ;: ;: . mum,mies CtS CtS 30 3 Q) MR S - - C/) C/) II . "C P E :E 20 I 2 a. E CtS ::J c: E CtS 10 1 c: Q) CtS Q) E E 0 0 13 27 11 25 8 22 5 19 3 17 Jun Jul Aug Sep Oct

Sample Date (1988)

Figure 3. Mean walnut aphids and I. pallid us mummies from the Colusa North Block. M= methidathion application, R=azinphosmethyl-resistant parasites released, P= applications of phosalone in alternate rows, E = ethephon application, S=parasites sampled for bioassays. Because methidathion had been applied to the block prior to parasite releases, no native parasites could be obtained for bioassays.

40 I - 1 4 - Q) ;: -Q) CtS ;:CtS 30 3 - C/) - . C/) "C 2 E :E 20 E a. ::J CtS E c: 1 c: CtS 10 CtS Q) Q) E E 0 0 13 27 11 25 8 22 5 19 3 17 Jun Jul Aug Sep Oct

Sample Date (1988)

Figure 4. Mean walnut aphids and I. pallidus mummies from the Colusa South Block. Parasitef were released (R) and native I. pallidus were sampled (S) prior to releases. Methidathion wa~ applied (M) to all rows, phosalone (P) was applied to alternate rows and ethephon (E) was applied. Parasites were sampled (S) for bioassays.

99 10 I I 1.0 A. Release trees 8; m aphids 1-0.8 mummies

:j S [060.4

R S -Q) -Q) ;;: ;;: 2 0.2 rn rn -Q) - en en 0 0.0 Q) 'tJ E .s:::.a. 10 1.0 E rn B. Non-release trees :J c: E rn c: Q) 8 0.8 rn E Q) E 6 0.6

4 0.4

2 0.2

0 0.0 4 18 2 16 30 13 27 11 25 8 22 Apr May Jun Jut Aug

Sample Date (1988)

Figure 5. Mean walnut aphids and I. callidus mummies from the Release (A) and Nonrelease (B) areas of the Gustine walnut block. Azinphosmethyl (A) was applied, parasites were released (R), and parasites were sampled (5) for bioassays.

100

-- -- Table 1. California walnut orchards where the azinphosmethyl-resistant strain of I. pallidus was released. Orchard Tree Date Size (acres) Pesticides applied 1988 County varieties #parasites R f\R Irriaation released block block

Hanford Vina 24 June 2.5 2.5 azinphosmethyl - 3 Ib (50WP)/250gaI/A - 9 June (3.36 kg 50WP/2338 I/hectare) Kings Chico 30,000 propargite -7.5 Ib (30W)/250 gallA, alternate rows - 29 July (7.51 kg 30W/2338 (/hectare) flood

Stockton Payne 23 June 3.0 2.0 azinphosmethyl - 2.2 Ib (35WP)/95 gallA - 24 May (2.46 kg 35WP/889 I/hectare) San Joaquin 9,000 azinphosmethyl - 2.0 Ib (50WP)/100 gallA - 9 July (2.24 kg 50WP/935 I/hectare) sprinkler ..... 0 ..... Colusa-North Serr 27 June 17 - chlorpyrifos - 3/4 gal (4E)/30 gallA - 11 March (7.02 I 4E/281 I/hectare) Glenn Vina 12,000 methidathion - 3/4 gal (2E)/120 gallA - 22 June (7.02 I 2E/1122 I/hectare) pull hose Chico phosalone - 4.5 pts (3E)/30 gal/A, alternate rows - 2 August (5.26 I 3E/281 IIhectare) Tehema phosalone - 4.5 pts (3E)/30 gall A, alternate rows - 18 August (5.26 I 3E/281 (/hectare) ethephon - 3.5 gal/35gallA - 29 August (32.74 , 2 (b/gal water-soluble liquid/327 I/hectare) - Colusa-South Serr 27 June 17 methidathion - 3/4 gal (4E)/120 gallA - 13-14 July (7.02 I 4E/1122 IIhectare) Glenn Vina 24,000 phosalone - 4.5 pts (3E)/30 gallA, alternate rows - 18 August (5.26 I 3E/281 IIhectare) pull hose Chico ethephon - 3.5 gal/35 gallA - 29 August (32.74 I 2 Ib/gal water-soluble liquid/327 I/hectare) Tehama Gustine Payne 23 June 2 1.5 azinphosmethyl - 3.5 Ib (50WP)/250 gallA - 16 June (3.92 kg 50WP/2338 I/hectare) Merced Chandler 1.800 flood Howard

...~ C t" Table 2. Survival rates of pre- and post-release colonies of I. pallidus collected from release (R) and nonrelease (NR) walnut blocks.

Date % survival after 24 h eXDosurea. Sample site collected ControlsJ:t 1988 R area NR area re-R Base Selected

Hanford pre-release 24 June 18 - - 16.1 0 94.0 post-release 14 July 3/1 81.1 80.0 22.9 0 92.5 post-release 11 Oct. 52/47 58.0 68.9 28.0 0 88.7

Gustine pre-release 23 July 28 - - 42.0 0 94.0 post-release 15 Aug. 6/- 26.4 - 34.0 14.1 90.0 Stockton pre-release 23 June 46 - - 0 0 94.0 post-release 7 July 16/- 72.5 - 3.0 0 77.5 post-release 12 Sept. 7/7 46.4 35.0 8.9 7.8 90.5 Colusa-Northblock pre-release 27 June 4 - - 2.0 0 60.0d post-release 16 Aug. 107 61.3 - 5.5 14.1 90.0 post-release 12 Oct. 53 52.0 - 11.1 5.7 85.5

Colusa-South block pre-release 27 June 46 - - 2.0 0 60.0d post-release 16 Aug. 46 36.5 - 5.5 14.1 90.0 post-release 12 Oct. .- 47 24.6 - 11. 1 5.7 85.5

a Ten parasites (both sexes) were tested with 50 ppm azinphosmethylwith a plastic cup bioassay at 250 C under 16L:8D for 24 h. b Controls were the azinphosmethyl-selected (Selected) and unselected (Base) colonies, as well as the colony (pre-R) collected from each site prior to the release of the azinphosmethyl-resistant strain. c The pre-release sample was obtained from the adjacent Colusa South block. d Growth chamber temperature was 270C rather than 250C.

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