# Effects of pesticide application and Neoseiulus barkeri on spider mite control using commercialized Neoseiulus californicus in a Japanese pear greenhouse

Yuya Mikawa, Mungunzaya Munkhtumur, Taichi Kawamura, Akio Yokoyama, Kotaro Mori, Masatoshi Toyama, Mineaki Aizawa, Shoji Sonoda

► To cite this version:

Yuya Mikawa, Mungunzaya Munkhtumur, Taichi Kawamura, Akio Yokoyama, Kotaro Mori, et al. Effects of pesticide application and Neoseiulus barkeri on spider mite control using commercialized Neoseiulus californicus in a Japanese pear greenhouse. Acarologia, 2025, 65 (3), pp.771-781. 10.24349/m6wg-dwoc. hal-05206329

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# Effects of pesticide application and *Neoseiulus barkeri* on spider mite control using commercialized *Neoseiulus californicus* in a Japanese pear greenhouse

Yuya Mikawa<sup>a</sup>, Mungunzaya Munkhtumur<sup>a</sup>, Taichi Kawamura<sup>a</sup>, Akio Yokoyama<sup>a</sup>, Kotaro Mori<sup>b</sup>, Masatoshi Toyama<sup>ID c</sup>, Mineaki Aizawa<sup>ID a</sup>, Shoji Sonoda<sup>ID a</sup>

## Original research

### ABSTRACT

A predator-release device with a waterproof shelter protecting a slow-release sachet containing *Neoseiulus californicus* (McGregor) (Acari: Phytoseiidae) has become commercially available for spider mite control. In this study, we examined the population dynamics of spider mites and phytoseiid mites in a Japanese pear greenhouse equipped with predator-release devices during 2019–2021. The presence of commercialized *N. californicus* on pear leaves before noticeable spider mite occurrence was confirmed during each of the survey years. Our results demonstrated that the spider mite density remained low for the first two years, but a notable spider mite outbreak occurred in the final year, presumably because a fungicide highly toxic to *N. californicus* was used before the release of the commercialized *N. californicus* individuals. Results also demonstrated that *Neoseiulus barkeri* Hughes (Acari: Phytoseiidae), the second dominant phytoseiid mite species, increased its relative proportion in the summer and possibly affected spider mite occurrence through intraguild predation. These results suggest that avoiding harmful pesticides and elucidating interactions among phytoseiid mite species are crucially important for more effective utilization of commercialized *N. californicus* for spider mite control.

**Keywords** biological control; microsatellite; phytoseiids; population dynamics; species identification

### Introduction

Spider mites are destructive pests of various crops, including Japanese pear (Hoy 2011; Van Leeuwen et al. 2015). The most notorious spider mite species in Japanese pear orchards is the two-spotted spider mite *Tetranychus urticae* Koch (Acari: Tetranychidae). Its outbreak causes scorching of leaves and precocious defoliation, thereby degrading fruit quality. Numerous and diverse pesticides have been developed for the control of *T. urticae*. Nevertheless, *T. urticae* has developed resistance to almost all the pesticides developed to date (Arthropod Pesticide Resistance Database, https://www.pesticideresistance.org/), due to its short life cycle and high reproductive potential (Van Leeuwen et al. 2015). Consequently, phytoseiid mites (Acari: Phytoseiidae), important natural enemies of spider mites (Helle and Sabelis 1985; Croft and

Received 31 January 2025

Accepted 18 July 2025

Published 05 August 2025

Corresponding author

Shoji Sonoda<sup>ID</sup>

sonodas@cc.utsunomiya-u.ac.jp

Academic editor

Kreiter, Serge

https://doi.org/10.24349/m6wg-dwoc

ISSN 0044-586X (print)

ISSN 2107-7207 (electronic)

<figure><img src="image_2.png" /></figure>

Mikawa Y. et al.

Licensed under
Creative Commons CC-BY 4.0

<footnote><sup>a</sup> School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.</footnote>

<footnote><sup>b</sup> Central Research Institute, Ishihara Sangyo Kaisha, Ltd., Kusatsu, Shiga 525-025, Japan.</footnote>

<footnote><sup>c</sup> Institute for Plant Protection, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8605, Japan.</footnote>
Jung 2001; van Lenteren 2001; Nomikou *et al.* 2002), have attracted attention for *T. urticae* control as alternatives to pesticide application in Japanese pear production.

The family Phytoseiidae includes more than 2,800 species (Demite et al. 2025). In Japan, ca. 100 species have been reported (Demite et al. 2025). *Neoseiulus californicus* (McGregor) (Acari: Phytoseiidae), classified as a type II specialist based on food utilization (McMurtry et al. 2013), has been used worldwide since 1985 as a biological control agent for spider mite control (Gerson and Weintraub 2007; van Lenteren 2012; Knapp et al. 2018). In 2017, a commercialized *N. californicus* release device named Miyako Banker® (ISK Bioscience K.K., Tokyo, Japan), hereinafter designated as a predator-release device, became available not only for greenhouse crops but also for outdoor vegetables and fruit trees, including Japanese pears (Mikawa et al. 2020, 2022). The device's waterproof shelter protects its slow-release sachet containing *N. californicus* from harsh environmental conditions such as high temperature, dryness, rainfall, and pesticide application (Banker-sheet®, ISK Bioscience K.K.). The device provides commercialized *N. californicus* with a refuge and a breeding place, facilitating their release over a long period (Mikawa et al. 2020, 2022).

Previously, predator-release devices were installed in a Japanese pear greenhouse to examine the population dynamics of spider mites and phytoseiid mites (Mikawa et al. 2020). Results demonstrated that commercialized *N. californicus* had dispersed to pear leaves by approximately one month after installation. However, a spider mite outbreak was observed in the summer, possibly because of the delayed settlement of released *N. californicus* on pear leaves. Results also revealed *N. californicus* as the most dominant species throughout the survey period, but the relative proportion of the second dominant species, *Neoseiulus barkeri* Hughes (Acari: Phytoseiidae) (*Neoseiulus makuwa* (Ehara) in the literature, see Discussion), increased in the summer (Mikawa et al. 2020).

This study was conducted to evaluate the effectiveness of predator-release devices for spider mite control and to elucidate factors correlating with spider mite outbreaks in the summer. For this study, predator-release devices were installed in the greenhouse to distribute commercialized *N. californicus* before noticeable spider mite occurrence. Thereafter, the population dynamics of spider mites and phytoseiid mites were examined during 2019–2021. To monitor commercialized *N. californicus*, a method to identify them was used: PCR-based selection of *N. californicus* from collected phytoseiids (Mikawa et al. 2019), followed by discrimination between commercialized and naturally occurring *N. californicus* using microsatellite markers (Mikawa et al. 2020).

# Material and methods

## Commercialized *N. californicus*

The predator-release device (Miyako Banker®; ISK Bioscience K.K.) consists of a waterproof shelter (Banker-sheet®; ISK Biosciences K.K.) holding a sachet of *N. californicus* (Biobest Belgium N.V., Antwerp, Belgium) (58 × 72 × ca. 5 mm), a black felt patch (trichome-mimicking material) (50 × 100 × 1 mm), and five water-absorbing polymers (moisturizing agents). The sachet contains *N. californicus* (ca. 100 individuals per sachet) and its prey, *Glycyphagus destructor* (Schrank) (Acari: Glycyphagidae), with bran as a bulking agent. The black felt patch and water-absorbing polymers within the shelter are expected to function respectively as oviposition and resting sites for *N. californicus* and humidity retainers (Shimoda et al. 2017, 2019). The manufacturer's recommendation for the installation was 100 predator-release devices per 1,000 m².

## Study site

Population surveys of phytoseiid mites and spider mites were conducted during 2019–2021 in a commercial Japanese pear greenhouse with a polyvinyl chloride plastic cover. The greenhouse
had windows on the ceiling that opened and closed automatically, with manually operated windows on the north and south sides. Japanese pear trees were trained on overhead horizontal trellis material, with a canopy height of ca. 1.8 m in the greenhouse. The exact age of each tree is unknown, but they were all more than 2.5 m tall; all produced fruits for sale. At the time of fruit thinning, the leaves of the lateral branches partially touched adjacent pear trees. No noticeable difference in the growth rates of Japanese pear trees was observed during the three surveyed years (data not shown). Several old trees in the greenhouse were replaced with younger trees before the 2019 and 2020 growing seasons. The greenhouse size (ca. 3,800 m²) of 2019 and 2020 was reduced to ca. 3,500 m² in 2021 because of conversion to grape production. In total, 400 predator-release devices were installed in the greenhouse on March 28, 2019 and March 25, 2021. In 2020, 200 predator-release devices were installed on March 26 and another 200 on April 30. One to three predator-release devices were placed around the trunks at ca. 1.0 m above the ground, depending on the tree size. Wild ground cover vegetation in the greenhouse was managed approximately once a month using a glyphosate herbicide (Roundup; Monsanto Co., Creve Coeur, MO, USA) and a mowing machine during the cultivation period each year. The pesticides highly toxic to *N. californicus* among those used in the greenhouse are presented in Table 1 (Table S1 provides all pesticides used) (Yoshimura *et al.* 2022). The pesticide types and their application schedules at the study site did not change to any considerable degree after introduction of the predator-release devices in 2017 (data not shown).

## Sampling procedure

Sampling was conducted in rows 1, 3, 5, 7, 9, and 13 out of the 13 rows from the entrance in 2019, in rows 2, 4, 6, 8, 10, and 12 out of the 13 rows from the entrance in 2020, and in rows 2, 4, 6, 8, 10, and 12 out of the 12 rows from the entrance in 2021. In each row, 15–20 trees were present. In each survey year, 20 leaves were collected randomly from four fixed trees in each row, i.e., a total of 480 leaves were collected for each sampling event. Sampling was conducted at 5-day to 14-day intervals during March 28 – October 30 in 2019, March 26 – October 28 in 2020, and April 5 – October 27 in 2021 (32 samplings in 2019 and 2020 and 28 samplings in 2021).

Leaves were brushed using a brushing machine (Daiki Co., Ltd., Konosu, Japan) to collect mites in each sampling event on a Petri dish filled with 70% ethanol. Using a binocular

<table><caption>**Table 1** Highly toxic pesticides to *Neoseiulus californicus* used at the study site during 2019–2021.</caption><thead><tr><th rowspan="2">Year</th><th rowspan="2">Date</th><th rowspan="2">Pesticide applied (Formulation, %AI)</th><th rowspan="2">IRAC mode of action classification</th><th rowspan="2">FRAC mode of action classification</th><th colspan="3">Toxicity category<sup>a</sup></th></tr><tr><th>Egg to adult</th><th>Adult female</th><th>No. of eggs oviposited<sup>b</sup></th></tr></thead><tbody><tr><td rowspan="3">2019</td><td>March 9</td><td>Diazinon (WP, 34.0)</td><td>1B</td><td>—</td><td>3</td><td>nd</td><td>nd</td></tr><tr><td rowspan="2">March 30</td><td>Cyanophos (WP, 40.0)</td><td>1B</td><td>—</td><td>1</td><td>nd</td><td>nd</td></tr><tr><td>Thiophanate-methyl (WP, 70.0)</td><td>—</td><td>1</td><td>1</td><td>1</td><td>4</td></tr><tr><td rowspan="3">2020</td><td>February 21</td><td>Cyanophos (WP, 40.0)</td><td>1B</td><td>—</td><td>1</td><td>nd</td><td>nd</td></tr><tr><td>March 17</td><td>Alanycarb (WP, 40.0)</td><td>1A</td><td>—</td><td>2</td><td>4</td><td>3</td></tr><tr><td>May 2</td><td>Thiophanate-methyl (WP, 70.0)</td><td>—</td><td>1</td><td>1</td><td>1</td><td>4</td></tr><tr><td rowspan="3">2021</td><td>February 25</td><td>Cyanophos (WP, 40.0)</td><td>1B</td><td>—</td><td>1</td><td>nd</td><td>nd</td></tr><tr><td rowspan="2">March 20</td><td>Benomyl (WP, 50.0)</td><td>—</td><td>1</td><td>3</td><td>1</td><td>4</td></tr><tr><td>Spirotetramat (FL, 22.4)</td><td>23</td><td>—</td><td>4</td><td>2</td><td>4</td></tr><tr><td rowspan="2">April 10</td><td rowspan="2"></td><td>Thiophanate-methyl (WP, 70.0)</td><td>—</td><td>1</td><td>1</td><td>1</td><td>4</td></tr><tr><td>Benomyl (WP, 50.0)</td><td>—</td><td>1</td><td>3</td><td>1</td><td>4</td></tr></tbody></table>

<footnote><sup>a</sup> IOBC/WPRS categorization of toxicities of pesticides based on their corrected mortalities: 1 (not harmful): < 30%, 2 (slightly harmful): 30–79%, 3 (moderately harmful): 80–99%, 4 (seriously harmful): 99% <.</footnote>

<footnote><sup>b</sup> Rate of decrease in the number of eggs oviposited (%) = 100 - [100 × (Mean numbers of eggs oviposited by adult females exposed to pesticide) / (Mean numbers of eggs oviposited by adult females exposed to water)] (Yoshimura et al. 2022).
nd: No data.</footnote>
microscope (SZY7; Olympus Corp., Tokyo, Japan), phytoseiid mites were separated from spider mites. Until DNA extraction, phytoseiid mites separated in each sampling event were stored, irrespective of their sex and developmental stage, in a glass container (4 ml) that had been filled with 3-3.5 ml of 99.5% ethanol. In 2019 and 2020, phytoseiid mites collected in each sampling event were mixed and stored together, but in 2021, the phytoseiid mites collected in each sampling event were mixed for each survey tree and stored. All collected spider mites were determined to be *T. urticae* based on morphological characterization using the previously described microscope.

## DNA extraction

Genomic DNA was extracted from phytoseiid mites collected during the survey period in 2019 (974 individuals), 2020 (1,062 individuals), and 2021 (2,521 individuals) using PrepMan Ultra (Applied Biomaterials, Warrington, UK) or MightyPrep reagent (Takara Bio Inc., Kusatsu, Japan). Briefly, a single phytoseiid mite introduced into 10 µL of the reagent was incubated at 95 °C for 10 min and then at room temperature for 2 min. After centrifugation at 15,000 × g for 2 min, the supernatant was recovered as a DNA sample. For subsequent PCR amplification, 0.2-0.5 µL of the supernatant was used.

## PCR amplification for discrimination of phytoseiid mite species

In 2019 and 2020, ribosomal internal transcribed spacer (ITS) sequences were used to identify *N. californicus*, *N. barkeri*, *Neoseiulus womersleyi* Schicha, *Amblyseius eharai* Amitai and Swirskii, *Amblyseius swirskii* Athias-Henriot, and *Gynaeseius liturivorus* (Ehara) using PCR, with species-specific primer sets, as described by Mikawa *et al.* (2019). For samples collected in 2021, species identification was conducted for *N. californicus* and *N. barkeri*. Quick Taq HS DyeMix (Toyobo Co., Ltd., Osaka, Japan) or EmeraldAmp MAX PCR Master Mix (Takara Bio Inc.) was used for PCR. The PCR conditions were the following: 1 cycle of 3 min at 94 °C, followed by 40 cycles of 15 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, with final extension at 72 °C for 7 min.

## Microsatellite genotyping and data analysis

Genomic DNA extracted individually from adult *N. californicus* females (417, 278, and 432 individuals, respectively, in 2019, 2020, and 2021) was used for nuclear microsatellite genotyping. Also, 24 naturally occurring samples collected in the greenhouse in 2017 and commercialized *N. californicus* samples reported by Mikawa *et al.* (2020) (35, 30, and 75 samples in 2019, 2020, and 2021, respectively) were used as references for the analyses. Adult *N. californicus* females collected in 2019, 2020, and 2021 were divided respectively into 18, 11, and 18 subpopulations, fundamentally based on the sampling date or month as presented below.

2019: April (April 24 and April 29), May 8, May 15, May 22, May 29, June 5, June 12, June 19, June 26, July 3, July 10, July 17, July 24, August 14, August 19, August 30, September 4, and September 11;

2020: May 7, May 13, May 20, May 27, June 3, June 10, June 17, June 24, June 30, July 8, and July 15;

2021: April 12, April 19, April 26, May 12, May 26, June 2, June 9, June 18, June 25, July 7, July 14, July 20, July 28, August 4, August 11, August 18, August 25, and September/October (September 22, October 6, October 13, October 20, and October 27).

Multiplex PCR using five nuclear microsatellite loci described in an earlier paper (Mikawa *et al.* 2020) was conducted using a PCR kit (Type-it Microsatellite; Qiagen Inc., Hilden, Germany). The PCR conditions were the following: 1 cycle of 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 90 s at 63 °C, and 30 s at 72 °C, with final extension for 30 min at 60 °C. The amplified PCR products were analyzed using a DNA sequencer (3500 Genetic Analyzer;
Applied Biosystems). Genotypes were determined using software (GeneMapper v.4.1; Applied Biosystems).

The number of alleles ($N_A$), observed ($H_O$) and expected ($H_E$) heterozygosities, and the fixation index ($F_{IS}$) were calculated using GenAlEx v.6.503 (Peakall and Smouse 2006). The deviation of $F_{IS}$ from the Hardy-Weinberg equilibrium was tested using FSTAT v.2.9.3.2 (Goudet 2001). The frequencies of null alleles were estimated using Cervus v.3.0.7 (Kalinowski et al. 2007).

Genetic structure was assessed using Bayesian clustering analysis implemented using InStruct (Gao *et al.* 2007) because inbreeding, along with deviation from the Hardy-Weinberg equilibrium and the presence of linkage disequilibrium, were observed, as described herein in *Results*. The results of 10 independent chains (runs) at $K = 2$, on which naturally occurring and commercialized *N. californicus* were mostly assigned to different clusters (Mikawa *et al.* 2020), were integrated using CLUMPP v. 1.1 (Jakobsson and Rosenberg 2007). Commercialized and naturally occurring samples were mostly assigned, respectively to Clusters 1 and 2 (data not shown). The results were presented as vertical bar plots (Excel; Microsoft Corp., Redmond, WA, USA).

## Statistical analysis

To evaluate the effects of *N. californicus* and *N. barkeri* on spider mite occurrence, a generalized linear mixed model (GLMM) with Poisson distribution and Gauss-Hermite quadrature method was constructed using the “glmmML» package in R ver.4.3.2 (R Core Team 2023). The number of spider mites was used as the response variable. The number of *N. californicus* and the number of *N. barkeri* were used as fixed effects. The trees from which leaves were collected were used as random effects. Analyses were conducted using data on each sampling date showing prominent spider mite occurrences (July 7 – August 18) in 2021.

## Results

### Occurrence of spider mites

The seasonal occurrences of spider mites and phytoseiid mites in the greenhouse during 2019–2021 are presented in Figure 1. Spider mite occurrence remained low throughout the survey periods in 2019 and 2020. In 2021, large populations of the spider mite were present during July–August, peaking in mid-August. The survey in 2021 showed that the seasonal occurrence of spider mites and phytoseiid mites varied greatly among survey trees (Figure 1).

### Species composition of phytoseiid mites

Genomic DNA extracted from 974, 1,062, and 2,521 phytoseiid mites collected respectively in 2019, 2020, and 2021, were examined for species identification using PCR. The most dominant species for the study period was *N. californicus* (62.4%, 61.5%, and 63.1%, respectively in 2019, 2020, and 2021), followed by *N. barkeri* (23.9%, 24.7%, and 31.4%, respectively in 2019, 2020, and 2021). Other phytoseiid such as *A. eharai* and *N. womersleyi* were less abundant in 2019 (0.5% and 1.0%, respectively) and 2020 (1.0% and 0.1%, respectively). The DNA samples showing no PCR amplification for any of the primer sets were treated as unknown.

The seasonal changes in phytoseiid mite species composition from 2019 to 2021 are shown in Figure 1. *Neoseiulus californicus* constituted a large proportion of the population throughout the survey period. *Neoseiulus barkeri* increased its proportion in the summer. This proportion increase of *N. barkeri* was evident in trees showing no noticeable appearance of phytoseiid mites in response to the spider mite occurrence such as tree numbers 2-1, 4-3, 6-2, 6-3, 8-1, 8-2, 8-3, 10-2, 10-3, 12-3, and 12-4 (Figure 1).
<figure><img src="image_3.png" /><figcaption>**Figure 1** Seasonal fluctuations in the species composition of phytoseiid mites (adults and immatures of both sexes) (bar) per leaf and in the numbers of spider mites (adults and immatures of both sexes) (line with filled circle) per leaf in the greenhouse during 2019–2021. Bars colored red, blue, and white respectively represent *Neoseiulus californicus*, *Neoseiulus barkeri*, and other phytoseiid/unknown samples.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2** Results obtained at *K* = 2 of Bayesian clustering analysis implemented in InStruct using five nuclear microsatellite loci for *Neoseiulus californicus* collected at the study site during 2019–2021.</figcaption></figure>

**Genetic structure of *N. californicus***

The characteristics of the five microsatellite markers used for this study (*N*<sub>A</sub>, *H*<sub>O</sub>, and *H*<sub>E</sub>) are listed in Table S2. Deviation from the Hardy-Weinberg equilibrium (*P* < 0.05) was detected for all five loci in 2019 and 2020, and for loci other than the NC019 locus in 2021. Linkage disequilibrium was observed between NC002 and NC013 loci in 2019, between NC019 and NC030 loci in 2020, and between NC002 and NC013 loci and between NC030 and NC048 loci in 2021, possibly because of the Wahlund effect of commercialized *N. californicus*, as reported for an earlier study (Mikawa *et al.* 2020).

The genetic structures of *N. californicus* collected between 2019 and 2021 are shown in Figure 2. *Neoseiulus californicus* individuals mostly assigned to Cluster 1 (commercialized *N. californicus*) first appeared on April 24, 2019, May 7, 2020, and April 12, 2021. In 2019, commercialized *N. californicus* was frequently observed until early July. Commercialized *N. californicus* was less prominent after mid-June 2020. The *N. californicus* individuals observed in May 2021 were far fewer than those observed in May 2019 and 2020.

**Effects of *N. californicus* and *N. barkeri* on spider mite occurrence**

The coefficients of *N. barkeri* in the GLMM analysis were greater than zero (*P* < 0.05) on most sampling dates (July 7, 14, 20, and 28 and August 4 and 18) in 2021 (Table 2), indicating that *N. barkeri* positively affected spider mite occurrence. By contrast, spider mite occurrence was
positively affected by *N. californicus* only on July 28, 2021 (Table 2).

<table><caption>**Table 2** GLMM analysis to examine effects of *Neoseiulus californicus* and *Neoseiulus barkeri* on spider mite occurrence in 2021.</caption><thead><tr><th rowspan="2">Sampling date</th><th colspan="2">Neoseiulus californicus</th><th colspan="2">Neoseiulus barkeri</th></tr><tr><th>Coefficient (SE)</th><th>P</th><th>Coefficient (SE)</th><th>P</th></tr></thead><tbody><tr><td>2024-07-07</td><td>-0.106 ± 0.166</td><td>5.25.E-01</td><td>0.437 ± 0.148</td><td>3.05.E-03</td></tr><tr><td>2024-07-14</td><td>-0.064 ± 0.202</td><td>7.51.E-01</td><td>0.210 ± 0.063</td><td>8.98.E-04</td></tr><tr><td>2024-07-20</td><td>-0.158 ± 0.088</td><td>7.27.E-02</td><td>0.689 ± 0.174</td><td>7.37.E-05</td></tr><tr><td>2024-07-28</td><td>0.171 ± 0.077</td><td>2.63.E-02</td><td>0.359 ± 0.156</td><td>2.09.E-02</td></tr><tr><td>2024-08-04</td><td>0.020 ± 0.020</td><td>3.04.E-01</td><td>0.147 ± 0.037</td><td>5.78.E-05</td></tr><tr><td>2024-08-11</td><td>-0.008 ± 0.022</td><td>7.17.E-01</td><td>0.099 ± 0.053</td><td>6.38.E-02</td></tr><tr><td>2024-08-18</td><td>0.082 ± 0.054</td><td>1.28.E-01</td><td>0.834 ± 0.195</td><td>1.81.E-05</td></tr></tbody></table>

Discussion

In this study, the PCR-based method described by Mikawa *et al.* (2019) identified species of high proportions (86.4%–94.5%) of collected phytoseiid mite individuals. Results of these analyses showed that the primer set developed for *N. makuwa* identification (Mikawa *et al.* 2019) was also applicable for *N. barkeri* identification (data not shown). Based on the nucleotide sequences of the amplified PCR fragments and morphological characterization, the phytoseiid mites identified as *N. makuwa* in our earlier papers (Mikawa *et al.* 2019, 2020, 2022) were *N. barkeri* (data not shown). Consequently, the specificities of the primers designed for phytoseiid mite species identification must be examined further. Nevertheless, the PCR-based method ascertaining the discrimination of phytoseiid mite species irrespective of their sex and developmental stage might be useful for researchers who lack adequate morphological identification skills or nucleotide sequencing systems at their institutions.

Our earlier paper reported that *T. urticae* occurs noticeably in May and that an approximately one-month period after installing the predator-release devices is necessary for the dispersal of commercialized *N. californicus* to pear leaves in the greenhouse (Mikawa *et al.* 2020). Therefore, for this study, predator-release devices were installed at the end of March. Consequently, commercialized *N. californicus* were distributed successfully on pear leaves before the noticeable *T. urticae* occurrence during 2019–2021 (Figures 1 and 2). In 2019 and 2020, the spider mite density remained low during the survey periods. However, a severe *T. urticae* outbreak occurred in 2021. In 2019 and 2020, many phytoseiid mites, with *N. californicus* as the dominant species, appeared in response to the *T. urticae* occurrence in May. In contrast, phytoseiids first appeared in late April and subsequently declined in May 2021. The *N. californicus* collected in May 2021 were far fewer than those collected during either of the prior two years (Figure 2). Benomyl, which is highly toxic to *N. californicus*, notably in egg production, was applied immediately before (April 10) commercialized *N. californicus* release in 2021 (April 12) (Table 1). No highly toxic pesticides were applied shortly before or after the commercialized *N. californicus* distribution in 2019 (April 24) or 2020 (May 7). Therefore, insufficient suppression of *T. urticae* density by commercialized *N. californicus*, followed by naturally occurring *N. californicus*, might have affected the *T. urticae* outbreak in 2021. These results suggest that avoiding the use of harmful pesticides is crucially important for more effective utilization of commercialized *N. californicus* for spider mite control.

*Neoseiulus californicus* and *N. barkeri* are classified, respectively, as selective predators of tetranychid mites (type II) and generalist predators (type III) based on their food utilization (McMurtry *et al.* 2013). *Neoseiulus californicus* preys on *T. urticae* even if the spider mite
colony produces heavy webs. On the other hand, type III generalists, including *N. barkeri*, show no adaptation to such spider mite colonies with heavy webbing (McMurtry *et al.* 2013). Consequently, the efficacy of *N. barkeri* in the suppression of *T. urticae* might be inferior to that of *N. californicus* in the greenhouse.

The survey conducted during 2019–2021 revealed *N. californicus* as the most dominant phytoseiid mite species, followed by *N. barkeri*, in the greenhouse (Figure 1). However, *N. barkeri* increased its proportion in the summer (Figure 1). The positive effects of *N. barkeri* on *T. urticae* occurrence were indicated by the GLMM analysis, rather than *N. californicus* (Table 2). To understand this result, we considered intraguild predation between *N. californicus* and *N. barkeri*. Intraguild predation, which occurs when two species share a host or prey, often engenders intense trophic interactions with subsequent high mortality among predators (Rosenheim *et al.* 1995). This phenomenon might exert a minimal effect on the shared prey population and might potentially hinder biological control efforts. Tsuchida *et al.* (2022) reported that the presence of *A. eharai*, type III generalists, diminishes phytoseiid complex effectiveness, potentially increasing pest numbers even when high-quality food is available in *Citrus natsudaidai*. Adult *N. barkeri* females that preyed on *N. californicus* larvae laid eggs, but adult *N. californicus* females that preyed on *N. barkeri* larvae did not lay eggs (Momen and Abdel-Khalek 2021). In the presence of *T. urticae*, adult *N. barkeri* females preyed on *N. californicus* larvae at an almost equal rate to *T. urticae* females, whereas *N. californicus* females preyed on *T. urticae* in preference to *N. barkeri* larvae (Momen and Abdel-Khalek 2021). Considering the characteristics of *N. barkeri* as a predator mentioned above, an increased relative proportion of *N. barkeri* might have negatively affected the predatory efficacy of *N. californicus*, thereby leading to the outbreak of *T. urticae* in the summer. To verify this hypothesis, intraguild predation among phytoseiid mite species, including *N. californicus* and *N. barkeri* should be examined in future studies.

Reportedly, various abiotic factors, such as extreme temperatures, solar ultraviolet-B radiation, low relative humidity, and pesticides, affect the survival, development, and fecundity of phytoseiid mites (Ghazy *et al.* 2016). In general, phytoseiid mites exhibit lower heat tolerance than spider mites, limiting their effectiveness in spider mite control under extremely high temperature conditions (Coombs and Bale 2013, Tscholl *et al.* 2022). In addition, low relative humidity is also known to hinder the potential of phytoseiid mites as biological control agents (Stenseth 1979). Effects of high temperature and relative humidity on the efficacy of predator-release devices also need to be examined for successful spider mite control in the future.

## Acknowledgements

This study was financially supported by the Ministry of Agriculture, Forestry, and Fisheries, Japan, through the Science and Technology Research Promotion Program for the Agriculture, Forestry, Fisheries, and Food Industry (28022C). The Japan Society for the Promotion of Science (JSPS) and The Japanese Government (MEXT) provided financial support to YM and MM, respectively.

## ORCID

Masatoshi Toyama ID https://orcid.org/0000-0003-0102-3266

Mineaki Aizawa ID https://orcid.org/0000-0002-8772-3855

Shoji Sonoda ID https://orcid.org/0000-0001-7683-0236
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