Phytoseiid predatory mites can disperse entomopathogenic fungi to prey patches

Recent studies have shown that predatory mites used as biocontrol agents can be loaded with entomopathogenic fungal conidia to increase infection rates in pest populations. Under laboratory conditions, we determined the capacity of two phytoseiid mites, Amblyseius swirskii and Neoseiulus cucumeris to deliver the entomopathogenic fungus Beauveria bassiana to their prey, Frankliniella occidentalis. Predatory mites were loaded with conidia and released on plants that had been previously infested with first instar prey clustered on a bean leaf. We examined each plant section to characterize the spatial distribution of each interacting organism. Our results showed that A. swirskii delivered high numbers of conidia to thrips infested leaves, thereby increasing the proportion of thrips that came into contact with the fungus. The effect was larger when thrips infestation occurred on young leaves than on old leaves. Neoseiulus cucumeris delivered less conidia to the thrips infested leaves. These patterns result from differences in foraging activity between predatory mite species. Amblyseius swirskii stayed longer on plants, especially within thrips colonies, and had a stronger suppressing effect on thrips than N. cucumeris. Our study suggests that loading certain predatory mite species with fungal conidia can increase their capacity to suppress thrips populations by combining predation and dispersing pathogens.


Results number and proportion of B. bassiana conidia delivered by predatory mites. The number of
CFUs recovered from the entire plant significantly differed among treatments (generalized linear model with negative binomial distribution, treatment χ 2 = 36.75, p < 0.001, Fig. 1). Both A. swirskii (multiple comparisons with 'glht' function, Tukey method, z = 6.45, p < 0.001) and N. cucumeris (z = 5.37, p < 0.001) contributed to increase total CFUs on plants compared to control. Both predatory mites delivered the same quantity of CFUs to the plant (z = 1.09, p = 0.519). One plant from the control treatment was excluded from the analysis because extremely high number of conidia (~19,800) landed on a single leaf; this outlier was more than three times of absolute deviation above the median 23 .
There was an interaction between the thrips oviposition leaf and treatment (generalized linear model with negative binomial distribution, interaction χ 2 = 31.47, p < 0.001; Fig. 2). Simple effects (the effect of each independent variable within each level of the other independent variable) were examined. For A. swirskii, this effect was greater when thrips eggs were laid on the young leaf than on the old leaf (generalized linear model with negative binomial distribution, χ 2 = 5.29, z = 2.32, p = 0.020). Amblyseius swirskii increased the number of CFUs recovered from the thrips oviposition leaf compared to control, but N. cucumeris did not (Kruskal-Wallis test, when thrips eggs were laid on old leaf: treatment simple effect χ 2 = 19.81, p < 0.001; Kruskal-Wallis test, when thrips eggs were laid on young leaf: treatment simple effect χ 2 = 18.55, p < 0.001).
At the beginning of the experiment, it was not possible to load the two predatory mite species with a similar number of conidia. Therefore, the proportion of B. bassiana delivered to the thrips oviposition leaf by the two predatory mite species was evaluated. The proportion of CFUs recovered from the thrips oviposition leaf varied among treatments (generalized linear model, χ 2 = 23.00 p < 0.001; Fig. 3) with A. swirskii increasing the proportion of B. bassiana on the thrips oviposition leaf compared to control (generalized linear model, followed by multiple comparisons with 'glht' function, Tukey method, z = 4.14, p < 0.001), but not N. cucumeris (z = 0.06,   (control) and with predatory mites, N. cucumeris or A. swirskii. Thrips oviposition leaf refers to the leaf where thrips females were caged for 24 hours to lay eggs prior to treatments. Dots identify outliers as defined by ggplot, i.e. values exceeding 1.5 interquartile range. Different capital and lower case letters indicate significant treatment effect for young and old leaf, respectively (p < 0.05, Kruskal-Wallis test with multiple comparisons). The asterisk indicates a significant difference (0.05 < p < 0.01) between thrips oviposition leaf: n.s. = not significant (Kruskal-Wallis test within treatment 'control' and treatment 'cucumeris' , generalized linear model with negative binomial distribution within treatment 'swirskii').  (control) and with predatory mites, N. cucumeris or A. swirskii. Different letters indicate significant differences between treatments (p < 0.05, generalized linear model with normal distribution, followed by multiple comparisons with 'glht' function, Tukey method). Dots identify outliers as defined by ggplot, i.e. values exceeding 1.5 interquartile range. p = 0.998). Amblyseius swirskii delivered a significantly higher proportion of B. bassiana to the thrips oviposition leaf than N. cucumeris (z = 4.15, p < 0.001).
proportion of thrips contacting B. bassiana delivered by predatory mites. The proportion of thrips coming into contact with B. bassiana was significantly affected by both treatment and thrips oviposition leaf (generalized linear model with normal distribution, treatment χ 2 = 22.37, p < 0.001; oviposition leaf χ 2 = 10.78, p = 0.001; Fig. 4), as well as by their interaction (χ 2 = 8.00, p = 0.018). For A. swirskii, this effect was much greater when thrips laid eggs on the young leaf rather than the old leaf (generalized linear model with normal distribution, followed by multiple comparisons with 'glht' function, Tukey method, z = 3.03, p = 0.002).
predatory mites and thrips remaining on the plant. Forty-eight hours following predatory mites released, higher numbers of A. swirskii (8.94 ± 0.88, mean ± S.E.) were recovered from the plants than N.

Discussion
Our results demonstrate that A. swirskii and N. cucumeris both have the capacity to disseminate B. bassiana conidia on plants when foraging. However, A. swirskii is more efficient than N. cucumeris as they delivered a higher proportion of conidia to thrips colonies.
There are mainly two ways in which conidia can be dislodged from the predatory mite body and be dispersed on the plant. They can either be actively groomed off by mites or rubbed off on the plant surface when predatory mites move along (Lin et al., unpublished data). Grooming, the use of legs to clean the body, has been observed in phytoseiid mites when they encounter potentially pathogenic fungi 24,25 . However, grooming is not efficient to remove all conidia from a mite, especially those located on the dorsal sections of their body. We further showed that A. swirskii and N. cucumeris mostly dislodged conidia from their body by walking on the plant surface. Indeed, the duration of walking is correlated to conidia removal for both species (Lin et al., unpublished data). Trichomes and other structures associated with the surface of bean leaves are likely to facilitate the dislodgement of conidia when mites are walking (Fig. 6B). Foraging predatory mites thus actively disperse B. bassiana conidia in the environment.
When mediated by predatory mites, transfer of conidia to thrips can either be a passive or an active process. Conidia are unloaded on plant surfaces and can subsequently passively attach to thrips cuticle when they forage www.nature.com/scientificreports www.nature.com/scientificreports/ on a contaminated substrate. Alternatively, conidia can be directly transferred from predatory mites to thrips during an unsuccessful predation event involving a physical contact between the two protagonists. Thrips are defensive prey that display counterattack behaviours. They can swing their abdomen to 'slap' predatory mites 26 or secrete irritating anal fluid, which causes predatory mites to withdraw 27 . Moreover, the presence of predatory mites in the vicinity of thrips colonies can affect their behaviour 28 . Following detection of predators, thrips may switch state from stationary feeding to escaping, thereby increasing the probability of coming into contact with spores disseminated on plant surfaces 14 .
The observed differences between A. swirskii and N. cucumeris in their capacity to disseminate B. bassiana conidia to thrips colonies might arise from differences in predator foraging patterns. Amblyseius swirskii is classified as subtype III-b generalist predatory mite adapted to mostly living on glabrous leaves, whereas N. cucumeris is classified as subtype III-e generalist predatory mite from soil/litter habitats 29 . As a consequence, more A. swirskii individuals were recovered from the plant than N. cucumeris at the end of our experiment. Such a pattern was also observed on cucumber plants infested with thrips 30 . However, habitat preference cannot alone justify differences in spore dispersal patterns between the two predatory mite species since they both deliver similar numbers of conidia to the plant. Amblyseius swirskii, a more efficient predator 30,31 , seems to be better adapted to detect thrips colonies and subdue this type of prey than N. cucumeris, as shown by A. swirskii suppressing thrips more strongly than N. cucumeris in our experimental setup. The difference in proportions of conidia delivered to thrips patches by the two predatory mite species attests to the better capacity of A. swirskii to exploit thrips on bean plants. In another study system 32 , showed that the predatory mite Neoseiulus (Amblyseius) barkeri Hughes (Acarina: Phytoseiidae) did not increase B. bassiana transmission to thrips. Neosiulus barkeri is a less voracious thrips predator than N. cucumeris with a relatively low capture success when attacking first and second instar larvae of Thrips tabaci Lindeman (Thysanoptera: Thripidae) 26 . Furthermore, the level of foraging activity of N. barkeri is lower than that of N. cucumeris 30 . These results suggest that the foraging capacity of a predator and the strength of its interaction with a prey would be essential determinants of its potential efficiency as a dispersal agent of entomopathogens.
The rate at which B. bassiana is contacting its host is crucial in the context of biological control, not only because it is directly linked to the infection rate but also because the viability of conidia is very sensitive to environmental conditions such as UV and humidity 33,34 . Typically, entomopathogens are used like pesticides with single or multiple applications of large quantities of pathogens in crops. However, in some instances, aerial applications are not effective to reach target pests. For example, due to its thigmokinetic behaviour, Western flower thrips are often concealed in plant crevices and flower buds 35 . As a result, spraying fungal pathogens has little effect on thrips infection level 36 . In such circumstances, the capacity of predatory mites in delivering pathogens to thrips colonies could increase disease transmission. In our experiment, the thrips mortality after contacted with pathogens was not evaluated. Nevertheless, it was shown that the LD50 is relative low for technical grade powder of B. bassiana: approximately 50 conidia per 2 nd instar F. occidentalis larva and only 5 per adult 37 .
Our findings about the relative potential of A. swirskii and N. cucumeris in dispersing B. bassiana conidia to thrips are consistent with the conclusion drawn by Zhang et al. 18 who studied a similar biological system on the tropical shrub, Murraya paniculata (L.) Jack (Rutaceae), infested by the Asian citrus psyllid, Diaphorina citri, in the laboratory. Higher mortality in D. citri populations was achieved when B. bassiana was delivered by A. swirskii rather than by N. cucumeris, and compared to B. bassiana being sprayed evenly onto plants. We can thus conclude that under our experimental conditions, A. swirskii is a better biological control agent because it reduced thrips more strongly and transmitted conidia to a larger number of thrips escaping from predation. However, N. cucumeris could show good potential both as a predator and an entomopathogen dispersal agent when used in a different crop-pest association. For example, in greenhouses from temperate regions, it has been shown that N. cucumeris showed similar performance as A. swirskii as a thrips biocontrol agent under simulated winter conditions 38 .
Finally, how can we apply such a system in a biological control program? Growers periodically release predatory mites and spray B. bassiana onto crops to control thrips. The strategy we proposed does not require two separate applications, but solely a premix of B. bassiana conidia (technical grade powder) into commercially available predatory mite package (if approved by regulatory agencies) 20 . The predatory mites would likely increase disease transmission rate to concealed pests. The overall quality of a predatory mite species as a pathogen dispersal agent would depend on its capacity to be loaded with conidia, its capacity to resist pathogenic infection and, as shown by the present study, its foraging activity. Predatory mites should be closely associated to the target pest and have the ability to search for, locate and engage in interactions with the pest on the plant, so they can disperse spores on the plant like little pebbles strewn about by Tom Thumb 39 . Methods the study system. The biological system under study consisted of the entomopathogenic fungus Beauveria bassiana, two species of predatory mites Amblyseius swirskii and Neoseiulus cucumeris as potential fungal dispersal agents and the western flower thrips Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) as a resource for both the fungus and the predators. These species share similar habitats (i.e. plants supporting thrips populations) and can coexist in commercial greenhouses applying biological control programs. In a previous study, we showed that B. bassiana strain ANT-03 is virulent to thrips (all stages, except first instar larva), slightly virulent to N. cucumeris and avirulent to A. swirskii 20 . This system thus perfectly fits the profile of a suitable pathogen, vector and host association, in which the pathogen is virulent against host and benign towards the vector 40 .
Beauveria bassiana is a generalist entomopathogenic fungus that exploits more than 200 species from most insect orders, with some isolates showing a high degree of specificity 41,42 . Conidia are responsible for infection and natural dispersal by air movement because of their small size (1-3 μm) 43 , by contact with infected hosts or via a dispersal agent 1,14,19 . Conidia adhere to the host cuticle, germinate, penetrate in the host by enzymatic (2019) 9:19435 | https://doi.org/10.1038/s41598-019-55499-8 www.nature.com/scientificreports www.nature.com/scientificreports/ and mechanical processes and next reproduce by exploiting host hemolymph and various host tissues [44][45][46] . Once host nutrients are depleted, the fungus breaches the cuticle from inside out and sporulates in large numbers 45 . Commercial strains of B. bassiana are used for the control of arthropod pests in biological control programs. They are typically sprayed over the crops like pesticides and the probability of contact with the host depends on the spatial distribution of the pests 36,42 .
The two phytoseiid species are generalist predators that actively search for prey 29 . Foraging phytoseiid mites typically respond to chemical cues emitted by plants when attacked by herbivores and move towards infested areas 47 . They are both commercialized and successfully released on vegetable and ornamental crops to control insect pests, including thrips 29 . They both mostly attacked first instar thrips larvae because larger prey successfully counterattack predatory mites 27 . Small and large thrips larvae live together in colonies on plant parts and larger larvae can protect their younger siblings from predation 48 .
Frankliniella occidentalis is a cosmopolitan and highly polyphagous insect that feeds on almost every plant parts, from leaves to flower and pods [49][50][51] , it can also vector a number of plant virus 52 . Their eggs are laid in plant tissues and then go through three stages (two larval and one prepupal stages) before pupation 52 . Frankliniella occidentalis can hide in concealed parts of plants where pesticides cannot reach them, and they rapidly develop resistance to chemicals 52,53 .

Arthropod colonies and fungal inoculum. A colony of N. cucumeris, provided by Anatis Bioprotection
Inc., was maintained on a factitious prey Aleuroglyphus ovatus Toupeau (Acari: Acaridae) while A. swirskii, purchased from BioBest Canada, was reared on a diet mixture containing Carpoglyphus lactis L. (Acari: Carpoglyphidae) and cherry pollen (Firman Pollen Co., Yakima, WA). Frankliniella occidentalis was obtained from a lab colony in Anatis Bioprotection Inc. and reared on California red kidney bean plants Phaseolus vulgaris L. (Fabaceae), with cherry pollen supplied ad libitum on a weekly basis. All colonies were maintained at 25 °C, 60-70% relative humidity and under a 14 L: 10D light cycle.
Beauveria bassiana strain ANT-03 has been registered in North America for greenhouse thrips control. We used the technical grade powder produced by Anatis Bioprotection Inc. containing 5 × 10 10 conidia g −1 for all experiments.
prey patch establishment on a plant. To test the capacity of predatory mites to deliver B. bassiana to thrips, we first established a spatial structure combining plant parts infested or not by thrips. To standardize the structure of a plant, we first trimmed bean plants (approximately 20 cm in height) to two sets of trifoliate (Fig. 7). To create a clumped distribution of thrips larvae on the plant, we enclosed 25 ovipositing female thrips for 24 hours in a clip cage on a single leaf. The clip cage was designed by F. Longpré, London Research and Development Center, Agriculture and AgriFood Canada, and made using a 3D printer. During the oviposition period, female thrips were assumed to have fed and left olfactory cues on the leaf that can further be used by predatory mites to locate the prey patch 54,55 . To avoid potential experimental bias related to leaf age or position, half of the plants had thrips on leaflet 2, the middle leaflet of the old trifoliate, while the second set of plants had thrips on leaflet 5, the middle leaflet of the young trifoliate (Fig. 7). Following oviposition, the clip cage and female thrips were removed from the plant. Four days later, when most eggs had developed into first instar larvae, the suitable prey stage for predatory mites, we released predatory mites loaded with B. bassiana on the plant.
Releasing predatory mites loaded with B. bassiana conidia. Adult female predatory mites of various ages were exposed to B. bassiana conidia in the commercial rearing substrate (2.5 × 10 9 conidia g −1 substrate) for two hours to obtain maximum conidia load on their body (Fig. 6) 20 . In a modified Eppendorf tube, we put 25 predatory mites with 0.2 g of B. bassiana contaminated rearing substrate 20 . The tube was attached on the stem, at equal distance to the base of the petiole of the two trifoliates (Fig. 7). To control for dispersal of B. bassiana conidia by air and potential mechanical disturbance during experimental manipulations, a tube containing 0.2 g of B. bassiana contaminated rearing substrate was attached to the plant (Control treatment). Each plant was www.nature.com/scientificreports www.nature.com/scientificreports/ isolated in a paper cylinder and the inner walls and the bottom of the paper cylinder were coated with rings of Tanglefoot ® glue to prevent conidia and predatory mites dispersal between experimental units. For each set of plants (leaf 2 vs. leaf 5), there were three treatments: control, B. bassiana dispersed by N. cucumeris and B. bassiana dispersed by A. swirskii. The experiment was repeated nine times (temporal blocks, n = 9) with two plants per treatment in each block at 25 °C, 60-70% relative humidity and under a 14 L: 10D light cycle. The two blocks where no thrips left on plants were excluded from the analyses of proportion of thrips bearing B. bassiana because this parameter cannot be estimated in absence of thrips.
Recovery of predators and prey. Forty-eight hours after the release of phytoseiid mites, plants were carefully examined to establish the number and spatial distribution of surviving predators and prey. Each of the nine plant parts (Fig. 7) were collected and placed in a 2 oz black solo cup with lid. The cup was filled with carbon dioxide from SodaStream ® to stop movement of thrips and predatory mites for the ease of handling and to avoid fungal cross-contamination between individuals. The number of mites and thrips found alive on each plant part was recorded. Thrips mortality was assumed to result from the presence of predators since B. bassiana conidia cannot germinate and invade thrips tissues within a 48 h period 20,56 .

Recovery of B. bassiana conidia from prey and plant parts.
To detect the presence or absence of B. bassiana on living thrips that remained on plants until the end of the experiment, thrips were individually picked with a sterilized toothpick or clean fine brush (sterilized with 75% ethanol and rinsed with 0.1% Tween-80 between samples) and placed in a small Petri dish (Ø 35 mm) containing 2.5 ml of an oatmeal selective media for B. bassiana 57 . Petri dishes were examined 10 days later when colony-forming units (CFUs) can be visualized. The proportion of thrips bearing conidia was calculated.
To assess the number of conidia on each plant part following arthropod removal, leaves and stems were cut into small pieces (<2 cm in width or length) and put back into the solo cup. Conidia were washed off by adding 5 ml of 0.1% Tween-80 into each solo cup and the cups were put on a rotary shaker for 2 hours at a speed of 125 rpm 58 . Next, one aliquot of a 0.5 ml suspension was transferred onto the selective media for B. bassiana 57 and CFUs were counted 9 days later. For each plant, we noted the sum of CFUs delivered to the entire plant and, more specifically, the quantity and the proportion of CFUs on the leaf where thrips females laid their eggs.
Statistical analyses. Our experimental design includes two categorical factors: treatment (3 levels: control, N. cucumeris and A. swirskii) and leaf where eggs were laid (2 levels: leaf 2 and leaf 5). When either factor was not identified as a significant predictor of the dependent variable following a log-likelihood test, it was eliminated from the initial statistical model to optimize the final model. The number of predatory mites remaining on the plants was analyzed using generalized linear models with negative binomial distribution and with species as a factor. The number of thrips remaining on the plants was analyzed with generalized linear models with negative binomial distribution and with treatment a factor. The proportion of thrips bearing B. bassiana was analyzed using generalized linear models with normal distribution with treatment and oviposition leaf as factors. The number of conidia delivered to the entire plant was analyzed with generalized linear models with negative binomial distribution and with treatment and oviposition leaf as factors. The number of conidia on the thrips oviposition leaf was analyzed with both generalized linear models with negative binomial distribution and Kruskal-Wallis tests, depending on whether the residuals were normally distributed or not, determined by Normal QQ-plot 59 . The proportion of conidia on the thrips oviposition leaf was analyzed with generalized linear models. Multiple comparisons were performed with the package 'multcomp' with 'glht' function and Tukey's all-pair comparisons method. Kruskal-Wallis multiple comparison tests were performed to compare differences among means when residuals were not normally distributed. All the statistical analyses were carried out with R version 1.0.143 60 .

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).