Mikawa, Yuya¹ ; Munkhtumur, Mungunzaya² ; Kawamura, Taichi³ ; Yokoyama, Akio⁴ ; Mori, Kotaro⁵ ; Toyama, Masatoshi ⁶ ; Aizawa, Mineaki ⁷ and Sonoda, Shoji ⁸

¹School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
²School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
³School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
⁴School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
⁵Central Research Institute, Ishihara Sangyo Kaisha, Ltd., Kusatsu, Shiga 525-025, Japan.
⁶Institute for Plant Protection, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8605, Japan.
⁷School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.
⁸✉ School of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan.

2025 - Volume: 65 Issue: 3 pages: 771-781

Original research

Keywords

Abstract

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 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 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 phytoseiids 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).

Genetic structure of N. californicus

The characteristics of the five microsatellite markers used for this study (N_A, H_O, and H_E) 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).

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.

acarologia_4816_supplementary_Table S2.xlsx

acarologia_4816_supplementary_Table_S1.xlsx

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