Sources of Mites and Thrips

Western flower thrips used in all tests were from a colony reared in our laboratory (University of Massachusetts), started about 1997 with material from a laboratory colony from Texas A & M University (K. Heinz laboratory). Thrips were reared on excised bean leaves by the method of Doane et al. (1995) modified by Lim et al. (2001). Neoseiulus cucumeris mites were purchased from Koppert Biological Systems (Koppert B. V., The Netherlands), shipped in bran with grain mites (Tyrophagus putrescentiae [Schrank], Acarina: Acaridae) and quality of these mites was consistently high, with large numbers of live mites in containers. Neoseiulus cucumeris mites were used in experiments immediately upon receipt. Iphisieus degenerans, packed in sawdust, were purchased from Biobest N. V. (Westerlo, Belgium). Shipments of I. degenerans repeatedly arrived in poor condition with many mites being dead or moribund. Before being used in experiments, live mites were placed on castor bean (Ricinus communis L., Euphorbiaceae) leaves dusted with red apple pollen and allowed to feed for at least 16 h to ensure that only mites in good health were used in experiments.

Experiment #1: Toxicity to Adult Thrips

Toxicity of spinosad at the full labeled rate (0.1198 g a.i./ml solution) to adult and larval thrips was tested as a positive control in view of the low toxicity that we expected to find for the beneficial mites. For thrips, we examined only the effects of 2 h-old residues.

For tests with adult thrips, excised leaves of young red kidney bean (Phaseolus vulgaris L., Fabaceae) plants produced in growth chambers were sprayed to the point of run-off with a hand held sprayer. Control leaves were sprayed with tap water. The nozzle of a hand powered spray bottle was held approximately 10 cm from leaf surfaces and a total of five sprays were applied to each leaf surface. Leaves were then allowed to dry at room temperature on the lab bench for 2 h at which time 2 × 1.5 cm leaf rectangles were cut and placed individually in 1-dram glass shell vials (Kimble Glass, Inc, Vineland, NJ). Vial caps had ventilation holes cut in them, and a small piece of Nitex screening (03-95/33) (Sefar America, Inc., Briarcliff Manor, NY) was held in place by the perforated cap. A disk (6 mm dia) of filter paper (#3 Qualitative) (Whatman Limited, Kent, U.K.) cut with a standard office hole punch was moistened with tap water and added to each vial to provide humidity.

Five adult female thrips from our laboratory colony were placed in each of 46 pesticide-treatment and 46 water-treatment vials. Vials were placed on trays and held for 24 h in a growth chamber at 22°C on a light cycle of 16:8 (L:D), and about 30% RH. After 24 h, thrips were checked for mortality.

Experiment #2: Toxicity to Larval Thrips

Spinosad-treated red kidney bean leaves were prepared as described above for tests with adult thrips up to the point of completed air drying. At that point, whole leaves were fixed to a disc of white paper cut to fit inside a plastic 15 × 1.5 cm petri dish (Falcon, Becton-Dickinson, Franklin Lakes, NJ). The edges of the leaves were taped with scotch tape to the disc and a thin barrier of tangletrap sticky material (Tanglefoot Co., Grand Rapids, MI) was applied around the whole edge of the leaf to produce a thrips-proof border. This ensured that test animals were continuously confined on the treated surface, which was not possible for winged adults.

With a fine paint brush, 10 immature (first or second instar larvae) thrips from the UMASS colony were placed near the center of the treated bean leaf in each of 24 pesticide-treated and 25 water-treated petri dishes. Petri dishes then were placed on trays and incubated for 24 h in a growth chamber at 22°C on a light cycle of 16:8 (L:D), and about 30% RH. After 24 h, petri dishes were closely examined and the number of dead thrips larvae counted.

Experiment #3: Toxicity to Both Mite Species

Bean leaves were treated with spinosad following the same procedures as described for adults thrips and allowed to dry in a greenhouse for 2, 24, or 48 h to simulate the natural degradation of the material expected under conditions of greenhouse use. Spinosad was applied at full label rate (0.1198 g a.i./ml solution) for all tests. Leaves with aged residues were taped into petri dishes and a tangletrap barrier was created around the leaf as in the experiment with larval thrips. Unlike conditions in the thrips tests, in the mite tests a dusting of red apple pollen and a 6-mm disk of moistened filter paper were placed on the bean leaf to supply mites with food and water. In tests with N. cucumeris and I. degenerans, 10 mites (any motile stage) were added per petri dish. Petri dishes were stacked on trays and placed in a growth chamber and held at 22°C, with a photoperiod of 16:8 (L:D) and about 30% R.H. Numbers of petri dishes per treatment varied from 6 to 27, as indicated in Table 2. Water disks were remoistened twice during the 24 h incubation period. After 24 h, the arenas were inspected and the numbers of live, dead, or missing mites recorded.

Experiment #4: Repellency to Both Mite Species

This test was run with 2-h-aged residues of spinosad, applied at the full label rate (0.1198 g a.i./ml solution) to the leaves of red kidney bean, as described above. Freshly sprayed leaves were allowed to dry indoors for 2 h. A test arena was constructed by cutting a spinosad-treated leaf in half along one side of the mid-vein and then taping it to half of a water-treated control leaf cut down the opposite side. The two leaf halves were overlapped slightly and white glue used to join them seamlessly. On the surface formed by these joined leaf halves, tanglefoot was applied as a barrier to create a square arena (10 cm on a side) centered on the dividing line so that exactly half of the enclosed surface was treated and half untreated. To initiate the test, 10 mites (the same procedure for both mite species) were placed on the line between the treated and untreated leaf halves. The arena was then continually observed for 15 min and mites on the treated and untreated areas were counted every 3 min. The five resulting counts were then summed for an individual replicate, such that a complete lack of repellency would be indicated by a 25:25 division of mite-observations between the two parts of the arena. For N. cucumeris, the responses of 300 mites (30 replicates) were examined and for I degenerans, 240 mites (24 replicates) were observed.

Experiment #5: Effect of Spinosad on Mite Oviposition

Spinosad-treated red kidney bean leaves were prepared as in the toxicity experiments described above, and then leaf squares (2 × 1.5 cm) were cut from the treated leaves after 2 h drying time and placed individually in 1-dram glass shell vials, as above. Vial caps were cut for ventilation and screened with Nitex screening (03-95/33) (Sefar America, Inc., Briarcliff Manor, NY). A 6-mm dia piece of moistened filter paper was added to each vial to provide humidity, along with a sprinkle of red delicious apple pollen (Antles Pollen Supplies, Wentachee, WA). For replicates testing oviposition of I. degenerans, 10 first instar thrips from the UMASS colony also were added to each vial as an additional food source for this species only. For replicates with N. cucumeris, only pollen was provided, as this is a sufficient food source for reproduction in this species. With a moist paintbrush, one mite of the test species was placed in each vial.

Vials were then placed on trays and incubated for 24 h in a growth chamber at 20°C, with a 16:8 (L:D) photoperiod and 50-70% RH. After 24 h, the surface of the leaf square and the inside of the vial were examined for mite eggs. Each surviving mite was then transferred into a fresh vial with a new leaf square cut from a plant that had been treated at the start of the experiment. The new vial also contained a fresh water disc, a sprinkle of apple pollen and, for I. degenerans, 10 new first instar thrips. This process was repeated so that oviposition was measured for three consecutive 24 h periods after set up.

Statistical Analyses

For experiments 1, 2, and 3 (toxicity to adult thrips, larval thrips, and mites, respectively), we used 2-sample t-tests to compare rates of mortality for treated and control groups. For experiments 4 (repellency) and 5 (effect on oviposition), we used the 2-sample t-tests with the Proc Mixed procedure of SAS to assess effects on repellency or mite oviposition.

Experiments #1 and #2: Toxicity to Adult and Larval Thrips

Western flower thrips confined on 2-h-aged spinosad residues at the labeled rate (0.1198 g a.i./ml solution) for 24 h showed low survival: 3% vs. 99% in controls for adults (t = 84.7, P < 0.0001, df =78) and 12% vs. 96% for larvae (t = 34.9, P < 0.0001, df = 40).

Experiment #3: Toxicity to Mites

In contrast to thrips, which suffered low survival when exposed to spinosad, N. cucumeris survival was high (74%) on even the least aged reside (2 h) and not significantly different from survival of mites on the water-treated control (78%) (t = 0.59, P < 0.56, df = 35). Most of the mortality suffered by N. cucumeris in either treatment was due to mites becoming stuck on the tanglefoot barrier around the arena or not being found at the end of the test (Table 1).

Survival of I. degenerans on fresh residues (2 h) was 56.3%, compared with 72.9% for controls (t = 4.25, P < 0.0001, df = 46). For 24 h-residues, survival rates were not different (65% on spinosad-treated leaves and 68% on water-treated controls). As with N. cucumeris, most mites did not survive and died by becoming stuck in the tanglefoot barrier.

Experiment #4: Repellency of Spinosad to Mites (Both Species)

Neither mite species showed any avoidance of leaf surfaces with freshly dried spinosad (full label rate, 2-h-aged residue): N. cucumeris (mean 25.2 ± 4.6 mites per treated zone vs. 23.3 ± 4.3 on the water control zone, n = 30) (F = 1.06, P < 0.38, df = 4, 116) and I. degenerans (24.0 ± 4.9 mites per treated zone vs 24.9 ± 5.1 on the water control zone, n = 24) (F = 1.28, P < 0.29, df = 4, 92).

Experiment #5: Effect of Spinosad on Mite Oviposition (Both Species)

Exposure to freshly dried (2 h) spinosad residues reduced oviposition of N. cucumeris on days 2 and 3 after confinement on pesticide-treated foliage by 48 and 76%, from 1.0 and 0.7 eggs per day on d 2 and 3 for controls to 0.5 and 0.2 on spinosad-treated leaves (Table 2), with n = 22-25 females for the spinosad-treated and 32 for the water-treated leaves (treatment: F = 13.55, P < 0.0004, df = 1, 105; time: F = 5.83, P < 0.004, df = 2, 105; interaction: F = 3.50, P < 0.034, df = 2, 105).

Oviposition of I. degenerans was reduced on days 2 and 3 by 41 and 70%, from 1.0 and 0.8 eggs per day for controls to 0.6 and 0.3 for mites on spinosad-treated leaves (Table 2), with n = 44 females for the spinosad-treated and 54 for the water-treated leaves (treatment: F = 18.52, P < 0.0001, df = 1, 186; time: F = 3.13, P < 0.046, df = 2, 186; interaction: F = 3.91, P < 0.022, df = 2, 186).