Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers

Biological Control 20, 191–198 (2001) doi:10.1006/bcon.2000.0896, available online at http://www.idealibrary.com on

Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers M. Navajas,* ,† H. Thistlewood,* ,‡ ,1 J. Lagnel,* D. Marshall,‡ A. Tsagkarakou,* and N. Pasteur† *CBGP, Institut National de la Recherche Agronomique, Campus International de Baillarguet CS 30016, 34988 Montferrier Sur Lez, France; ‡Pest Management Research Centre, Agriculture & Agri-Food Canada, Vineland, Ontario, L0R 2E0, Canada; †Institut des Sciences de l’Evolution (CNRS, UMR 5554), Laboratoire Ge´ne´tique et Environnement, Universite´ Montpellier II, 34095 Montpellier, France Received August 5, 1999; accepted November 4, 2000; published online February 6, 2001

The predacious phytoseiid mite Neoseiulus fallacis (Garman) is an important agent for the biological control of spider mites in deciduous fruit orchards in North America and Canada. It would be helpful to monitor the fate of released individuals to improve the results of introductions of the predators in biological control trials. We have used two types of genetic markers, pyrethroid resistance and allozymes, for indirect estimation of the survival of N. fallacis introduced in an apple orchard in Ontario, Canada. Mite samples were submitted to toxicological tests. The polymorphism of four enzymes was studied in individual females using an isoelectric focusing technique. A mite sample was taken from the field, mass-reared in the laboratory, and selected for permethrin resistance. This strain was released on several apple trees treated with permethrin, and mite samples were collected from the same trees 10 to 90 days later. The genetic composition and the insecticide resistance level of this sample were compared to those of two other samples from trees where mites had not previously been released, either in the same orchard or in a neighboring block. A control susceptible strain was compared using mites collected earlier from trees on the same site but outside the present experiment. The mites collected from the release trees and those from the strain used for the releases were found to be genetically closely related, as judged from a small genetic distance, and from similar levels of insecticide resistance in both samples. The control samples from the nonrelease trees were genetically distant from these and displayed low resistance levels. These results indicate that the released genotypes established and persisted in the release trees for the period of the experiment. The utility of the two approaches in assessing the fate of released natural enemies is discussed. © 2001 Academic Press

1 Current address: Pacific Agri-Food Research Centre, Agriculture & Agri-Food, Canada, Summerland, B.C. V0H 1Z0, Canada.

Key Words: Neoseiulus fallacis; mites; releases; biological control; allozymes; toxicological tests; insecticide resistance; genetic similarity.

INTRODUCTION

Predation is a key factor influencing spider mite (Acari: Tetranychidae) outbreaks (Wilson et al., 1998). Neoseiulus fallacis (Garman) is an important indigenous predator for biological control of Tetranychus urticae Koch and Panonychus ulmi Koch (Tetranychidae) in several Canadian fruit crops. It also feeds on rust mites (Eriophyoidae), pollen, and honeydew (see Pratt et al., 1999). The use of pesticides and the subsequent elimination of phytoseiid mite predators (Thistlewood, 1991; Hill and Foster, 1998) and the absence of effective registered acaricides in high value crops have renewed interest in the augmentative and inundative release of phytoseiids for spider mite biological control in North America (Croft, 1990; McMurtry, 1982; Morris et al., 1996; Strong et al., 1997). The development of resistance to pesticides in mite predators offers excellent possibilities for integrating spider mite biological control with chemical control of pests (Van der Vrie, 1985). Currently, a mass-reared and pesticide-resistant strain of N. fallacis is being tested and released on several crops in Canada. Small-scale releases of N. fallacis have been performed in North America, with varied results attributed to factors including hybridization with native strains, dispersal rate, poor overwintering of released mites (Prokopy and Christie, 1992), and the use of single or mixed phytoseiid species (Strong and Croft, 1995). Questions concerning release rate, establishment success or failure, dispersal rate, and overwintering survival are being examined in Canada to determine the costs or benefits and to discover whether or not this approach has any long-term value. The avail-

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ability of markers to postrelease monitoring in the field would be decisive in assisting the introduction of natural enemies. In addition, knowledge of the fate of the released individuals might contribute to the evaluation of eventual indirect and nontarget effects of biological control programs (Cory and Myers, 2000). The outdoor release of mass-reared mite predators, and particularly inundative release, can provide equivocal results and requires an understanding of predator dispersal and population structure that has been only partially investigated (Dunley and Croft, 1992, 1994; McDermott and Hoy, 1997; Strong et al., 1999). Although genetic markers are important tools in characterizing arthropod movement, dispersal, and possible gene flow between introduced and native individuals, they have only been rarely applied for biological control purposes (see Hopper (1996) for a review on the factors making biological control effective). Insecticide resistance has been employed as a genetic marker for N. fallacis and other phytoseiids to provide information on their dispersal and establishment after release and was used to detect the survival of susceptible and resistant strains of N. fallacis in apple orchards in Michigan (Whalon et al., 1982), and to estimate dispersal of Typhlodromus pyri Scheuten in Oregon apple orchards (Dunley and Croft, 1994). Allozyme analysis has been employed on phytoseiid mites in studies of predation (Solomon et al., 1985; Fitzgerald et al., 1986; Dicke and de Jong, 1988), establishment and dispersal (Whalon et al., 1982; Dunley and Croft, 1994), and systematics (Messing and Croft, 1991), in particular using nonspecific esterase systems. A promising alternative source of genetic markers is the isoelectric focusing electrophoresis (IEF) technique, described for small arthropods by Kazmer (1991) and modified by Tsagkarakou et al. (1996) for T. urticae. This method allows the investigation of up to 4 to 5 enzyme systems for each mite, enabling a multilocus study of such small organisms (Tsagkarakou et al., 1997, 1998). In this paper, the technique is applied to the Phytoseiidae and the potential of IEF for providing polymorphic genetic markers is assessed for 45 enzymatic systems. We conducted an experimental study to monitor the results of the release of an organophosphate- and pyrethroidresistant strain of N. fallacis in an apple orchard in Ontario, Canada, receiving different spray programs. The fate of the releases was analyzed by two methods: (1) by determination of the degree of resistance to permethrin in the mites collected before and after the introduction of the resistant strain and (2) by isozyme genetic characterization of introduced and native mites.

5⬘N, 79° 32⬘W). A 3- to 5-year-old apple orchard with 39 rows (east to west), each with 61 trees at 3 ⫻ 5 m spacing, the northern half planted with cv. ‘Red Delicious’ and the southern half with cv. ‘McIntosh’, was bordered on all sides by a 10-m-wide track. In 1994, the entire orchard was treated with mineral oil (Superior Oil, NM Bartlett Inc., Beamsville, Ontario, Canada) applied at 60 L/ha in the dormant period on 20 April, with captan (Captan 80WP, Zeneca Agro, Stoney Creek, Ontario, Canada) at 1.4 kg active ingredient per hectare (kg AI/ha) on 10 May, 18 May, 25 May, 1 June, 24 June, and at 2 kg AI/ha on 8 July, 27 July, and 26 August. Similarly, in 1995, using the same materials and rates as in 1994, the orchard was treated with mineral oil in the dormant period on 5 May, and with captan at 1.4 kg AI/ha on 12 May, 26 May, 11 June, 26 June, and at 2 kg AI/ha on 10 July, 26 July, and 16 August. For our experiment, the orchard was divided into three areas, separated by guard rows 11 to 14, and 25 to 28, which were not sampled. The western 12 rows, designated area OP, received mainly organophosphate insecticides as recommended by the local extension service for commercial orchards, and in 1994, the treatments were permethrin (Ambush 500 EC, Zeneca Agro) at 0.12 kg AI/ha on 14 May, diazinon (Diazinon 50 WP, Novartis Crop Protection, Mississauga, Ontario, Canada) at 1.6 kg AI/ha on 7 June, phosmet (Imidan 50WP, Zeneca Agro) at 0.82 kg AI/ha on 17 June, and at 1.25 kg AI/ha on 7 July and 10 August. In 1995, they were diazinon on 1 June, phosmet on 16 June, 7 July and 8 August, azinphos-methyl (Guthion 50 WP, Bayer Inc., Toronto, Ontario, Canada) at 0.75 kg AI/ha on 26 July and 25 August, and methomyl (Lannate L, Dupont Canada, Toronto, Ontario, Canada) at 6.75 L/ha on 3 August. The central area (rows 13 to 26) received a “selective” program with only diflubenzuron (Dimilin 25WP) applied at kg AI/ha on 14 May and 16 August 1994, and 8 August 1995 and tebufenozide (Confirm 240 F, Rohm & Haas Canada, Mississauga, Ontario, Canada) at 0.14 L AI/ha on 17 June 1994 and 16 June 1995. The eastern third of the orchard (designated area P, rows 27 to 39) was treated with permethrin (Ambush 500 EC, Zeneca Agro) at 0.12 kg AI/ha on 14 May, 17 June, and 11 August 1994 and on 15 May, 16 June, and 8 August 1995. Chemicals were applied in the early morning, usually under windless conditions, using a Rittenhouse Model 328 W6 air-blast sprayer calibrated to deliver 336 L/ha (May to June) or 530 L/ha (July) by the tree row volume method.

MATERIALS AND METHODS

Mite Strains Experimental Orchard and Crop Protection The experiment was conducted at Agriculture & Agri-Food Canada’s Jordan Experimental Farm (43°

Five N. fallacis strains are described. A strain (MR) was created from N. fallacis collected from a commercial orchard, selected in the laboratory for permethrin

MONITORING FIELD RELEASES OF Neoseiulus fallacis

resistance (Thistlewood et al., 1992), and mass-reared (Applied Bio-Nomics Ltd., Sidney, British Columbia Canada) on kidney bean, Phaseolus vulgaris L, infested with two-spotted spider mite, T. urticae. The strain MR was used for the experimental releases. A strain (P8) was created from mites collected from older apple trees on the same site, but outside the present experimental orchard, and maintained in the laboratory as a standard “susceptible” strain (Thistlewood et al., 1992). A strain (OP) was formed from mites collected in the part of the orchard treated with organophosphate insecticide and forms a control outside the release zone. Strains PC and PR were from sampling performed in the orchard after the last release of MR: strain PC was from trees not infested with MR and PR was from trees infested with MR. Strains PC, PR, and OP were constituted from mites collected in the field and reared in the laboratory for a few generations to increase numbers. Strains MR and P8 had been reared in the laboratory since 1989. The MR strain was regularly supplemented with individuals recaptured from the field some time after release in order to preserve variability and selected with permethrin each generation. Release of N. fallacis and Recovery from Trees Mites of the strain MR were shipped in containers containing approximately 1000 N. fallacis on bean leaves with some prey. The leaves were divided and attached to the trees by plastic “twist-ties” at an estimated rate of 250 mobile stages per tree. Samples of leaves were set aside at random during the release and examined in the laboratory under a binocular microscope to determine the actual numbers released. Eighty trees from the middle section of the pyrethroid area were selected using random number tables. Ten trees per cultivar were designated as check (no release) trees and 30 trees per cultivar as predator release trees (the strains PC and PR, respectively, were sampled from these trees). N. fallacis was released on 21 June, 13 July, and 15 August 1995 in the PR trees. Phytoseiid mites were recovered from trees using sentinel plants (Whalon et al., 1982). Kidney bean plants, P. vulgaris, ca. 10 cm high with the root ball bagged in plastic, infested with one or two T. urticae females per leaf, were stapled in the center of trees from 24 August to 5 September and from 11 to 18 September 1995. Any mites were removed using a modified Berlese technique for 4 h or by hand under a microscope and phytoseiid mites were reared (Thistlewood et al., 1995). Representative mites were mounted regularly and identified using interference contrast microscopy and samples were verified by Eiko YoshidaShaul, Department of Zoology, University of Toronto, Toronto, Canada.

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Pesticide Bioassay Assay of the toxicity of permethrin for the different samples of N. fallacis was performed using a petri dish method (Thistlewood et al., 1992). Permethrin (technical grade, Zeneca Inc.) was dissolved in 95% ethanol and serial dilutions were performed to the appropriate concentrations for tests. Control dishes were treated with 95% ethanol. All concentrations are given as milligrams per liter of active ingredient on a weight to volume basis. For bioassays, mortality was assessed after 24 h and mites were considered dead if incapable of moving two steps after being prodded gently with a fine paintbrush. N. fallacis were held during assay or posttreatment in controlled environment chambers at 24 ⫾ 1°C, 60% RH, 16:8 L:D. Results were tabulated for a series of concentrations and concentration–mortality regression lines were computed by probit analysis using POLO-PC software (LeOra Software, Berkeley, CA), which incorporates Abbott’s correction for control mortality and ␹ 2 tests for the heterogeneity of expected and observed values of the regression line. Regression lines were compared for equality of slopes and intercepts and for parallelism by the likelihood ratio test. All values are expressed as the mean, x, ⫾ SE, and P ⫽ 0.05 level. Electrophoresis Four samples (OP, PC, PR, and MR) were studied in this analysis. Strain P8 had been reared for several years in the laboratory and was excluded from the genetic study, although it was compared for pesticide resistance. Predators were collected during the first to third generations of rearing postrecovery, placed in cryogenic tubes, and held at 4°C for at least 2 days to clear the gut before being frozen at ⫺70°C. Frozen samples were transported in dry ice and stored in liquid N 2 until electrophoresis. Isoelectric focusing on cellulose acetate membranes (Separax-EF, Fuji Photo Film, Tokyo, Japan) was performed. Individual adult females from each population were homogenized directly on the membrane in a line ca. 2 cm from the anode. At least 10 individual females from each sample, to a total of 90 per run, were placed on the top of a stack of three membranes impregnated with pH 4 –5.6 gradient carrier ampholytes (Pharmacia LKB; Uppsala, Sweden) and voltage was applied (500 V for 15 min, 1000 V for 15 min, 1250 V for 10 min, and 1500 V for 50 min). After migration, each membrane was used to reveal a different enzyme system (Pasteur et al., 1988). Preliminary tests to detect protein activity were performed for 45 enzyme systems (Pasteur et al., 1988) using homogenates from 5–10 mites each of the MR, P8 samples and some individuals of the spider mite T. urticae as positive control. Of these, four systems showed some polymorphism and

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TABLE 1 Concentration–Mortality Response to Permethrin in Samples of N. fallacis Assayed by the Petri Dish Method Sample a

N

LC 50 (mg/L)

95% CL

Resistance ratio

Slope ⫹ SE

␹2

df

P

P8 b OP PC PR MR

600 700 800 800 600

2.3 50.8 36.0 2397.0 8776.0

0.6–4.0 40.3–65.3 23.3–55.4 1692–3,957 6118–11,034

— 22 16 1042 3816

2.2 ⫹ 0.5 1.1 ⫹ 0.1 1.2 ⫹ 1.0 1.1 ⫹ 0.1 1.3 ⫹ 0.2

1.7 2.5 10.6 8.7 4.3

3 4 5 5 3

⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05

a b

See text for the description of the origin of each sample. From Thistlewood et al. (1992).

were selected for further genetic analysis: aspartate aminotransferase (Got, EC 2.6.1.1), phosphoglucomutase (Pgm, EC 2.7.5.1), esterases (Est, EC 3.1.1.1, 3.1.1.2), and glucosephosphate isomerase (Pgi, EC 5.3.1.9). For the best results, after electrophoresis the stacked membranes were stained in the following order from top to bottom: Got and Pgm (double-stained on one membrane), Pgi, and Est. The PC population was chosen as reference for naming alleles, the most common allele being named “100” and other alleles being named in relation to their isoelectric point relative to the 100 allele. Genetic Data Analysis Population structure was analyzed with GENEPOP version 3.1d software (Raymond and Rousset, 1995a). Deviations from the Hardy–Weinberg equilibrium (heterozygote deficit or excess) were determined by the Fisher exact test (Louis and Dempster, 1987). Genotypic linkage disequilibrium for all pairs of loci was tested in each sample and genetic differentiation between samples was tested by comparing the allelic composition of all pairs of samples for all loci by a Fisher exact test (Raymond and Rousset, 1995b). The significance level of each test was adjusted by taking into account the other tests, using the sequential Bonferroni method (Holm, 1979). Ohta (1982) indices of variance components of linkage disequilibrium were computed using the LINKDOS program (Garnier-Gere and Dillmann, 1992). To assess sample differentiation, estimates of F ST were computed according to Weir and Cockerham (1984). The genetic divergence between samples was estimated by the unbiased genetic similarity and distance coefficient (Nei, 1978) in a cluster analysis using the unweighted pair– group method with arithmetic means computed using BIOSYS Release 1.7 software (Swofford and Selander, 1981). RESULTS

Release of N. fallacis Three releases were performed in 1995 at the recommended rate employed for field tests in Ontario. Actual

densities of mobile stages released were 141 ⫾ 16.8 (SE) (21 June), 208.5 ⫾ 18.4 (SE) (13 July), and 201 ⫾ 24.7 (SE) (15 August) per tree. Eggs were counted but not reported. Pre- and postrelease samples (10 –50 leaves per tree) on 21 June, 28 June, 5 July, and 18 September showed very few (⬍0.015 per leaf) N. fallacis at any time. Tetranychid mite densities were high at the time of release, reaching 55 mobile stages per leaf in late June, and dropping to near zero in the last sample, with no significant differences observed between densities in control and release trees. Despite the low population density, N. fallacis were successfully recovered from the OP, PR, and PC trees and reared in the laboratory. Very few phytoseiid mites other than N. fallacis were found in the permethrintreated and nearby “commercial” OP areas. By contrast, in the nearby “selective” area the predators were almost all Neoseiulus andersoni (Chant) and too few N. fallacis were found for rearing or analysis. Insecticide Bioassays Results of the permethrin assays are given in Table 1. Values for the MR strain are from an assay prior to shipping the laboratory-selected N. fallacis to the mass-rearing insectary, after which the strain was treated with permethrin during each rearing cycle. The concentration–mortality responses for all samples were linear (P ⫽ 0.05 level). The LC 50 values of the susceptible reference P8 sample are typical of pyrethroid-susceptible N. fallacis collected in southern Ontario (H.M.A.T., unpublished data) and those of the released MR strain are ⬎3800 fold higher. Values for the field control OP and PC samples are slightly higher (16- to 22-fold) than those of the P8 reference and not significantly different from one another (95% CL of LC 50 are overlapping). By contrast, the resistance ratio of the PR sample was intermediate between PC and MR (e.g., ⬇1000 fold) and was typical of that found only in association with releases of the MR strain (H.M.A.T., unpublished results). These results indicated that N. fallacis of the release MR strain and mites recovered from the trees (PR) receiving the MR strain have significantly higher levels of resistance

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TABLE 2 Allelic Frequencies and Inbreeding Coefficient (F IS) in Samples of Neoseiulus fallacis from an Experimental Trial in Ontario, Canada Locus

Alleles b

OP a

PC

PR

MR

26 0 0 1 —

22 0 0 1 —

Got

N 80 90 100 F IS

35 0.186 0.014 0.800 0.048

32 0.125 0 0.975 0.441

Pgm

N 90 100 110 F IS

35 0.086 0.800 0.114 0.256

48 0.042 0.948 0.01 ⫺0.035

41 0.037 0.963 0 0.661

23 0.087 0.0891 0.022 ⫺0.078

N 80 90 100 120 F IS

36 0.111 0.153 0.722 0.014 ⫺0.116

49 0.224 0.102 0.653 0.020 0.213

42 0.643 0.167 0.190 0 0.036

29 0.638 0.362 0 0 0.102

Pgi

a b

See text for the description of the origin of each sample. Number of tested individuals.

than indigenous mites, exemplified by the strains OP and PC. Electrophoresis Among the 45 enzyme systems tested in preliminary tests, activity was observed only for Got, Pgm, Est, Idh, 6-phosphogluconate dehydrogenase (Pgd), glucose-6phosphate dehydrogenase (G6pd), and Pgi. Of these, the Got, Pgm, Est, and Pgi systems repeatedly showed strong activity and variations in electrophoretic mobility. Two loci encoding esterases were identified as a result of the hydrolysis of ␣- or ␤-naphthyl acetate but only one, Est-1, that was monomorphic could be scored. Between 22 and 52 individuals were analyzed per locus and per sample. Fasting of the predators for 24 h to remove gut residues of T. urticae and prior tests enabled us to separate the alleles of T. urticae from those of N. fallacis in the four enzyme systems.

remain significant (P ⬎ 0.05) when multiple tests were taken into account. It can be concluded that the loci studied give independent information. Following the procedure of Ohta (1982), we investigated whether the linkage disequilibrium between alleles of each pair of loci was due to directional selection pressures or to genetic drift. For all pairs of loci, Ohta’s procedure indicated that the nonrandom genotypic associations found over the whole data set were most likely due to genetic drift rather than to a uniform selection pressure. Genetic Differentiation The Fst estimates were computed to investigate the differentiation in genotypic frequencies among samples. The overall differentiation among samples was significant F ST ⫽ 0.30 (P ⬍ 0.05). Results at the Got, Pgm, and Pgi loci generally agreed with one another, although the highest variability and degree of discrimination were observed at the Pgi locus. The genetic differentiation was also analyzed for pairs of samples (Table 3). The lowest F ST (0.018; P ⬎ 0.5) was found for the comparison between PC and OP and both samples were not significantly different. All other comparisons were significantly different (P ⬍ 0.05). However, comparison between samples PR and MR disclosed a relatively low F ST value (0.049; Table 3). A cluster analysis was performed between samples (Fig. 1) using the matrix of genetic distances computed between pairs of samples (Table 3). The mass-reared strain (MR) had been released in certain trees only and was genetically closest to the mites recovered from the release trees (PR). Similarly, the genetic distances were relatively small among populations from the same orchard but in trees not receiving the release (PC) as well as from trees at the same site (OP) that represented a control external to the pyrethroidtreated area employed for the release experiment. DISCUSSION

Assessment of the fate of introduced natural enemies can be helpful in selecting strategies for integrated

Genetic Polymorphism The allele frequencies observed for the three polymorphic loci in the five samples of our study are reported in Table 2. Heterozygote excesses and deficits were determined. None of the two excesses (F IS ⬍ 0) or the three deficits (F IS ⬎ 0) detected were significant (P ⬎ 0.16). The genotype linkage disequilibrium in each strain was tested to determine whether the polymorphism of each locus studied was independent of the others. A significant disequilibrium was observed only between Pgm and Pgi in sample OP (P ⬍ 0.001), which did not

TABLE 3 Genetic Differentiation among Samples of N. fallacis: Estimates of the F ST (lower diagonal) and Nei’s (1978) Genetic Distance (Upper Matrix)

OP PC PR MR

OP a

PC

PR

MR

— 0.018** 0.272* 0.322*

0.005 — 0.216* 0.306*

0.082 0.051 — 0.049*

0.113 0.085 0.008 —

Note. Significant genetic differentiation: *P ⬍ 0.05; **ns. a See text for the description of the origin of each sample.

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FIG. 1. Dendrogram of N. fallacis samples based on genetic distance (Nei, 1978). Cluster analysis was performed using the UPGMA algorithm.

pest management. In this study, we attempted to indirectly estimate the survival of N. fallacis introduced in one apple orchard in Ontario. We introduced this phytoseiid experimentally and monitored its fate after release in the field. We observed the same patterns of differentiation among mite populations, both by physiological bioassay of the toxicity of permethrin and by allozyme analysis. The strains formed by indigenous mites in the field (PC and OP) are genetically close and each very different from strain MR used for infestation. This difference was noted mainly for the loci Got and Pgi (Table 2). The strain MR is fixed for the Got 100 allele, whereas PC and OP have three alleles. The most frequent allele at locus PGI is not the same in MR (Pgi 80) as in OP or PC (Pgi 100). Unlike OP and PC, strain PR was collected in a part of the field in trees previously infested by MR. From the allele frequencies of the loci Got and Pgi, it would seem that PR is genetically closer to the released strain MR than to OP and PC. These genetic relations between strains are represented graphically by the dendrogram plotted using Nei’s genetic distances (Fig. 1). Two groups can be identified in Fig. 1: the strain (PR) from the trees infested with N. fallacis and the strain used for infestation (MR), and a second group consisting of the strains (PC and OP) sampled from trees in the same area but collected from trees not infested with MR. These results are in agreement with the data obtained by toxicological tests. On the one hand, the strains PC and OP display low, similar resistance to permethrin (LC 50 ⫽ 36 mg/L and LC 50 ⫽ 51 mg/L, respectively). On the other hand, strain PR displays much higher resistance (LC 50 ⬇2400 mg/L), more similar to that of MR (LC 50 ⬇8800 mg/L), which was selected by permethrin. These results can be interpreted in two ways. The first hypothesis is the introgression of strain MR by hybridization with indigenous mites. The lifetime of this species is approximately 40 days (Sabelis, 1985) and the collection of mites began 9 days to 2 months after the release of MR. It is likely that the MR mites had time to mate with the native strain of mites

or that a mixture of MR and indigenous individuals was collected as PR. Whatever the case, it is clear that the MR mites survived for several days to 2 months in the trees in which they were released and that at recapture these individuals, or their descendants, were capable of reproducing under rearing conditions to form strain PR. Detection of isoenzymatic polymorphism by isofocalization shows that these markers can be used for studies of the structure of populations of N. fallacis. Unfortunately, the absence of a diagnostic allele for the released strain MR makes accurate estimation of the strain in the field difficult. It is clear that the mites survived until recapture but our data do not show clearly whether they reproduced with an indigenous population in the field. It cannot be excluded from our protocol that the PR sample was in fact a mixture of released mites (MR) and mites that were originally in the field before the releasing procedures (OP). Knowledge of this proportion might allow the estimation of the proportion of released individuals from field samples or at least inform us to what proportions of released individuals in field samples may be detected. We attempted to infer this proportion based on the obtained genotypic data. We simulated field samples by combining individual genotypes from the OP data set and MR data set in different proportions in the range of 0 to 1% in a data set of 100 different combinations. We subsequently calculated the genetic distance between the simulated field samples and the MR strain. Knowing that the measured genetic distance between PR and MR is 0.008, the simulated data allow the extrapolation of the proportion of PR/MR that corresponds to this distance value. To obtain a genetic distance of 0.008, a proportion of 28% of OP should be contained in the MR sample. This rationale led us to conclude that sample collected 2 months after the releasing procedures contains 78% MR mites, the rest being either OP mites or descendents of OP and MR. An alternative method aimed at obtaining this type of information consists of rendering the released strain homozygotic for a genetic marker not present in the experimental field. Such a kind of genetic label will reliably trace the fate of the introduced mites. Likewise this technique will allow the estimation of how long the released individuals can be detected in the field, which will be a guide in designing further experimental protocols. At the time that releases were being tested in Ontario, the usual way of tracing the establishment of introduced predators was by counting the entire mite community and comparing numbers in release versus nonrelease trees or blocks. Such assessments have been notoriously flawed, particularly with respect to highly mobile species such as N. fallacis (Prokopy and Christie, 1992; Lester et al., 1999). The development and easy availability of a mass-reared pyrethroid-re-

MONITORING FIELD RELEASES OF Neoseiulus fallacis

sistant strain, MR, made it possible to monitor a release by using toxicological tests to examine resistance to pyrethroids. However, such tests require a large number of individuals, hence the need to rear the individuals recaptured after release. As an alternative, Whalon et al. (1982) made the first attempt to employ electrophoresis for analysis of released phytoseiids. The number of allozyme assays that can be performed on an individual mite is limited by the amount of protein that can be isolated and the number of available enzyme assays (Pasteur et al., 1988). The procedure is also less sensitive than current molecular tests and does not allow one to mark individuals or follow lines of descendants. Unfortunately, attempts to develop molecular monitoring tools, such as RAPD markers, have proven inconsistent or difficult for individual phytoseiid mites for a number of reasons (Perrot-Minnot and Navajas, 1995; Edwards et al., 1998), and a recent attempt for N. fallacis, using microsatellite sequences, proved similarly unfruitful (Navajas et al., 1998). However, the present study shows that the genetic monitoring of N. fallacis releases is now possible using relatively small numbers of individuals and leads to suggestions to increase effectiveness and accuracy. In particular, it would be desirable to (1) examine samples collected directly from the natural environment to eliminate problems related to rearing in the laboratory, (2) examine the genetic structure of the indigenous populations before release, and (3) perform sampling during recapturing operations at least 2 weeks after releases to detect possible introgression between the released strains and the autochthonous population. Sampling performed as few as several days after the last releases (as studied here) provides information about the survival of the mites released but not on the long-term establishment of the individuals introduced or on their interactions with any preexisting populations. ACKNOWLEDGMENTS We are grateful to C. Chevillon and G. Fauvel for valuable comments on the manuscript. We thank Don Elliott (Applied Bio-Nomics Ltd., Sidney, BC, Canada) and Noubar Bostanian (AAFC, Horticultural Research Centre, Quebec, Canada) for providing mite material, Darlene Nesbitt for assistance, and David Kazmer (University of Wyoming, WY) for materials. H.M.A.T. thanks Jean Gutierrez, Lloyd Knutson, and Franc¸ois Leclant for facilities and advice at the Zoology Laboratory INRA-ENSAM and European Biological Control Laboratory (USDA-ARS). Funding was provided in part by Energy, Mines, and Resources Canada, the Department of External Affairs, and an AAFC-USDA Cooperative Research Agreement. This is Contribution 2001002 of the Institut des Sciences de l’Evolution.

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