bioRxiv preprint doi: https://doi.org/10.1101/2020.01.11.901751; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Imidacloprid Movement into Fungal Conidia is Lethal to Mycophagous

ROBIN A. CHOUDHURY*, ANDREW M. SUTHERLAND*, MATHEW J. HENGEL, MICHAEL P. PARRELLA, and W. DOUGLAS GUBLER

*these authors contributed equally to the writing, design and completion of this work First author: University of Texas Rio Grande Valley, School of Earth, Environmental, & Marine Sciences; second author: University of California Cooperative Extension, Alameda County; third author: University of California, Davis Department of Environmental Toxicology; fourth author: University of Idaho, College of Agriculture and Life Sciences; fifth author: University of California, Davis Department of Plant Pathology.

Abstract. 1. Applications of systemic pesticides can have unexpected direct and indirect effects on nontarget organisms, producing ecosystem-level impacts. 2. We investigated whether a systemic insecticide (imidacloprid) could be absorbed by a plant pathogenic fungus infecting treated plants and whether the absorbed levels were high enough to have detrimental effects on the survival of a mycophagous . Beetle larvae fed on these fungi were used to assess the survival effects of powdery mildew and imidacloprid in a factorial design. Fungal conidia were collected from treated and untreated plants and were tested for the presence and concentration of imidacloprid. 3. Survival of beetles fed powdery mildew from imidacloprid-treated leaves was significantly lower than that of beetles from all other treatments. 4. Imidacloprid accumulated in fungal conidia and hyphae was detected at levels considered lethal to other , including coccinellid beetles. 5. Water-soluble systemic insecticides may disrupt mycophagous insects as well as other nontarget organisms, with significant implications for biodiversity and ecosystem function. Key words. Psyllobora vigintimaculata, Podosphaera xanthii, powdery mildew, imidacloprid, neonicotinoid, tri-trophic interaction, non-target effect.

Introduction Neonicotinoid insecticides, which disrupt nicotinic Systemic insecticides are considered valuable tools acetylcholine receptors, are commonly employed for pest management. Products containing water-soluble worldwide within pest management programs (Matsuda active ingredients are applied to roots via the substrate or et al., 2005). Imidacloprid, the first commercially to leaves and are readily translocated throughout the successful neonicotinoid, has excellent systemic plant. Active toxins accumulate in the plant tissue and properties. Imidacloprid products are labeled for use are toxic to feeding on the plants. Such against many phytophagous pests in agricultural and applications protect active ingredients from ultraviolet urban landscapes, such as piercing/sucking insects, bark light and other degradation factors, enabling them to burrowers, and chewing beetle larvae, but activity is persist longer in the environment. In addition, soil evident on a wide range of arthropods. Direct toxicity applications of systemic insecticides have sometimes has been shown not only to common target pests such as been considered less harmful than sprays to beneficial aphids and whiteflies (Qu et al., 2015; Smith et al., insects active in the plant canopy while specifically 2016), but also to beneficial insects such as coccinellid targeting phytophagous pests (Pflueger & Schmuck, beetles (Lee et al., 2017), hymenopteran parasitoids 1991). (Tappert et al., 2017), and predatory mites (Put et al., 2016). bioRxiv preprint doi: https://doi.org/10.1101/2020.01.11.901751; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A growing body of literature exists documenting Concurrently, pumpkin seeds were planted with the direct and indirect effects of imidacloprid and other Sunshine Mix potting medium 1 (SunGro Horticulture, neonicotinoids against pollinator insects (Easton & Bellevue, Wash.) into five 6-inch pots in trays and Goulson, 2013; Goulson, 2013). For instance, the flood irrigated with either 17.4% imidacloprid (EPA imidacloprid-laced pollen from seed-treated sunflowers Registration# 264-763-AA-67760) or deionized water was implicated in deaths of foraging honeybees and in a greenhouse. After ten days, one tray each of decreases in honey production in France (Bonmatin et imidacloprid-treated and untreated plants were removed al., 2003). Guttation fluids from treated plants have also from the greenhouse, inoculated with P. xanthii (by been identified as routes to exposure for nontarget gently transferring conidia from infected leaves of other arthropods (Hoffmann & Castle, 2012). Indirect plants), and placed into a growth chamber (25°C, 50% toxicity to beneficial insects may also readily occur. It RH). The other two trays were kept uninoculated in the has been shown that coccinellid predators may ingest greenhouse (20° ± 10°C, 50% ± 20% RH). lethal doses of imidacloprid through the consumption Fifteen days later, leaves from all four seedling of sessile homopteran prey that have ingested this groups were removed and cut petioles inserted into 2 systemic material through phytophagy (Grafton- ml glass vials with deionized water, sealed with Cardwell & Gu, 2003). Even consumption of parafilm. These vials were then placed into inverted honeydew from homopterans feeding on treated plants transparent plastic quart (~ 950 ml) containers and can negatively impact the fecundity and survival of sealed. One first- or second-instar beetle larva was beneficial insects (Calvo-Agudo et al., 2019). introduced to each container. Containers with Imidacloprid and its plant metabolites move readily uninfected leaves (without powdery mildew) were through various organisms at different trophic levels, supplemented with one 15 mm leaf disk cut from imparting toxicity to susceptible insects. Such trophic untreated infected plants, to provide a food source for movement violates the integration clause of integrated P. vigintimaculata larvae, known as obligate pest management. mycophages (Davidson, 1921). Excised leaves and Plant pathogenic fungi that utilize plant water and vials were changed every four days to prevent wilting; nutrients, such as the powdery mildews (Erysiphales), supplemental infected leaf disks were changed every may act as reservoirs for systemic insecticides, and three days. In this way, treatments included: 1) therefore may be toxic to susceptible arthropods if untreated and uninfected with infected leaf disc consumed. The cosmopolitan coccinellid tribe Halyziini supplement: IM-/PM-, 2) untreated and infected with is known to obligately consume Erysiphales conidia powdery mildew: IM-/PM+, 3) imidacloprid-treated and hyphae, providing ecosystem services in natural and uninfected, with infected leaf disc supplement: and agricultural settings throughout the world IM+/PM-, and 4) imidacloprid-treated and infected (Sutherland & Parrella, 2009). with powdery mildew: IM+/PM+. Each treatment was Using this model, we sought to determine whether represented by 15 replicate containers, for a total of 60 imidacloprid can move into fungal hyphae and whether bioassay arenas (see Figure S1). Containers were this movement would negatively affect beetles that fed maintained at ambient room temperature (22°C) and on the contaminated hyphae. 12h photoperiod. Mortality, moribundity, and developmental stage were assessed for beetle larvae Materials and methods daily until emergence of adults from pupae. Moribund larvae that were immobile for two or more days were Adults of the halyziine coccinellid beetle Psyllobora judged dead and removed from the assay. Observations vigintimaculata (Coleoptera: Halyziini) were collected continued until all larvae were dead or had successfully from a vineyard in Fresno, CA in fall 2011. Beetles pupated. Conidia from both imidacloprid treated and were reared in a growth chamber (PGR-15, Conviron untreated leaves were vacuum aspirated into a Ltd., Winnipeg, Canada) that was kept at 25°C and microcentrifuge tube and tested for imidacloprid 50% relative humidity under fluorescent lights with a concentration using liquid chromatography and tandem 14-h photoperiod on pumpkin plants (Cucurbita pepo mass spectrometry (see supplemental materials for cv. Sorcerer) infected with the cucurbit powdery specific methods used). mildew pathogen, Podosphaera xanthii (syn. Survival analyses were conducted in R v. 3.5.2 using Podosphaera fusca). To encourage a uniformly aged the ‘survival’ package (Therneau, 2015). To analyze population for our bioassay, we introduced the main effects of imidacloprid and powdery mildew approximately 400 beetles of mixed sex into a separate treatments on survival, in addition to the interaction growth chamber containing pumpkin plants infected ten effects, we generated both a full regression model and days prior. After four days of egg deposition, adults performed a pairwise logrank test using Bonferoni- were removed from the colony and egg broods left to adjusted p-values. hatch. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.11.901751; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Fig. 1: Survival of obligately mycophagous beetle larvae, P. vigintimaculata, after being confined to leaves from plants either treated or untreated with imidacloprid (IM+/IM-) and either infected or uninfected by powdery mildew fungus Podosphaera xanthii (PM+/PM-). Larvae confined to uninfected leaves (PM-) were regularly provided sustenance by way of supplemental leaf discs from infected and untreated plants.

Results Discussion Qualitative examination of survival curves suggests that This study represents the first record of the tri-trophic the treatment groups began to diverge by ~24h post movement of imidacloprid from plant to fungus to exposure (Fig. 1), and that 100% of beetles raised on insect, ultimately creating significant indirect mortality powdery mildew from imidacloprid-treated leaves died of a nontarget organism and potentially disrupting a before the end the study. Survival regression; including pathway for biological control of an important group of imidacloprid treatment, powdery mildew treatment, and pathogens. We observed that all beetle larvae fed on the interaction between the two as explanatory factors; powdery mildew grown on imidacloprid-treated plants revealed that the effects of imidacloprid treatment was perished within 120 hours. In contrast, larvae confined substantially higher [hazard ratio (HR)= 3.66, 95% to imidacloprid-treated leaf arenas but regularly confidence interval (CI): 1.86–7.18] than the effect of provided with leaf discs from untreated infected plants powdery mildew food availability (HR=2.18, 95% CI: exhibited no significant differences in survival as 1.13–4.19). We also found a strong interaction between compared to those fed on powdery mildew grown on the imidacloprid treatment and the presence of powdery untreated plants. These observations, as well as the mildew (HR=4.99, 95% CI: 1.23–20.21). survival regression results, suggest that the insecticide Chemical analysis detected increased levels of did not impact survival of larvae through volatilization imidacloprid in conidial samples taken from leaves from treated leaves nor through incidental phytophagy. excised from plants treated with imidacloprid. Powdery Instead, mortality was associated strictly with mildew conidia collected from leaves treated 15-days consumption of fungal structures growing from treated prior had imidacloprid concentrations of 33.6 µg/g plant tissue. This conclusion is supported by previous (n=3), whereas conidia from untreated leaves exhibited observations that soil-applied imidacloprid is readily concentrations below the detectable level (0.05 µg/g) translocated into various tissues of C. pepo plants (n=1) (Figure S2). Conidia collected from leaves 22 (Stoner & Eitzer, 2012) and that lethal effects of days after the imidacloprid treatment exhibited a mean imidacloprid consumption on three different coccinellid concentration of 7.65 µg/g (n=5), while untreated species has been observed at 6.03 µg/g (Krischik et al., leaves continued to have concentrations below the 2015). In general, imidacloprid is considered toxic to detectable level (n=4). coccinellid larvae, with lethal residues at concentrations as low as 2.6 µg/g (Mizell & Sconyers, 1992). bioRxiv preprint doi: https://doi.org/10.1101/2020.01.11.901751; this version posted January 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Mycophagous beetles may play an important role in References management and detection of powdery mildew disease, and reductions in their populations might exacerbate Bonmatin, J., Moineau, I., Charvet, R., Fleche, C., Colin, M., & Bengsch, E. (2003) A LC/APCI-MS/MS method for analysis of imidacloprid in soils, in powdery mildew disease outbreaks in susceptible crops. plants, and in pollens. Analytical Chemistry, 75, 2027-2033. Calvo-Agudo, M., González-Cabrera, J., Picó, Y., Calatayud-Vernich, P., Urbaneja, Sutherland and Parrella (2006) found that a single A., Dicke, M., & Tena, A. (2019) Neonicotinoids in excretion product larvae of P. vigintimaculata removed an average of 6.3 of phloem-feeding insects kill beneficial insects. Proceedings of the National Academy of Sciences, 116, 16817-16822. cm2 of leaf area affected by powdery mildew during Davidson, W. (1921) Observations on Psyllobora taedata LeConte, a coccinellid development from egg to adult. Furthermore, Peduto et attacking mildews. Entomol. News, 32, 83-89. Easton, A.H. & Goulson, D. (2013) The neonicotinoid insecticide imidacloprid al. (2011) found that incidence of P. vigintimaculata repels pollinating flies and beetles at field-realistic concentrations. adults was positively correlated with incidence of early PLoS One, 8, e54819. Goulson, D. (2013) An overview of the environmental risks posed by neonicotinoid season grapevine powdery mildew disease, suggesting insecticides. Journal of Applied Ecology, 50, 977-987. Grafton-Cardwell, E.E. & Gu, P. (2003) Conserving vedalia beetle, Rodolia a possible use of P. vigintimaculata as a bioindicator. cardinalis (Mulsant) (Coleoptera: ), in citrus: a The use of mycophagous beetles in greenhouse settings continuing challenge as new insecticides gain registration. Journal of Economic Entomology, 96, 1388-1398. to directly consume powdery mildew or as early Hoffmann, E.J. & Castle, S.J. (2012) Imidacloprid in melon guttation fluid: A indicators of disease may help direct management of potential mode of exposure for pest and beneficial organisms. Journal of Economic Entomology, 105, 67-71. diseases in such controlled settings (Parrella & Lewis, Krischik, V., Rogers, M., Gupta, G., & Varshney, A. (2015) Soil-applied 2017). Mycophagous beetles from the Halyziini are imidacloprid translocates to ornamental flowers and reduces survival of adult Coleomegilla maculata, Harmonia axyridis, and Hippodamia observed throughout the world in association with convergens lady beetles, and larval Danaus plexippus and Vanessa cardui butterflies. PloS one, 10, e0119133. powdery mildew infections (Sutherland & Parrella, Lee, Y.S., Jang, M.J., Lee, H.A., & Lee, J.H. (2017) Toxicity of pesticides to 2009), suggesting that the potential for indirect mycophagous ladybird, koebelei Timberlake (Coleoptera: Coccinellidae: Halyziini). Korean Journal of Pesticide Science, 21, mortality of mycophagous beetles after exposure to 364-372. systemic insecticides is widespread. Matsuda, K., Shimomura, M., Ihara, M., Akamatsu, M., & Sattelle, D.B. (2005) Neonicotinoids show selective and diverse actions on their nicotinic The ecosystem-level impacts of direct and indirect receptor targets: Electrophysiology, molecular biology, and receptor exposure to lethal doses of systemic insecticides is still modeling studies. Bioscience, Biotechnology, and Biochemistry, 69, 1442-1452. being explored. Several studies have explored the tri- Mizell, R.F. & Sconyers, M.C. (1992) Toxicity of imidacloprid to selected predators in the laboratory. Florida Entomologist, 75, 277- trophic movement of systemic insecticides and the 280. subsequent disruption of natural predators and Parrella, M.P. & Lewis, E. (2017) Biological control in greenhouse and nursery production: Present status and future directions. American parasitoids (Calvo-Agudo et al., 2019; Grafton- Entomologist, 63, 237-250. Cardwell & Gu, 2003; Lee et al., 2017; Put et al., 2016; Peduto, F., Sutherland, A., Hand, E., Broome, J., Parikh, P., Bettiga, L., Smith, R., Mahaffee, W., & Gubler, W. (2011) Comparing the efficiency of Tappert et al., 2017). While some studies demonstrated visual scouting, spore trapping systems and a bioindicator for early the lethal impacts of direct applications of pesticides to detection of Erysiphe necator in California vineyards. Phytopathology, 101, S139-S139. mycophagous beetles (Lee et al., 2017; Sutherland et Pflueger, W. & Schmuck, R. (1991) Ecotoxicological profile of imidacloprid. Pflanzenschutz-Nachrichten Bayer, 44, 145-158. al., 2010), our study showed that indirect exposure to Put, K., Bollens, T., Wäckers, F., & Pekas, A. (2016) Non‐target effects of imidacloprid through the fungal food source can also commonly used plant protection products in roses on the predatory mite Euseius gallicus Kreiter & Tixier (Acari: Phytoseidae). Pest rapidly lead to death in mycophagous beetles. Management Science, 72, 1373-1380. Widespread measurements of reductions in insect Qu, Y., Xiao, D., Li, J., Chen, Z., Biondi, A., Desneux, N., Gao, X., & Song, D. (2015) Sublethal and hormesis effects of imidacloprid on the soybean diversity and abundance have recently been attributed aphid Aphis glycines. Ecotoxicology, 24, 479-487. to intensive agricultural activities and associated Sánchez-Bayo, F. & Wyckhuys, K.A. (2019) Worldwide decline of the entomofauna: A review of its drivers. Biological Conservation, 232, 8- pesticide inputs (Sánchez-Bayo & Wyckhuys, 2019); 27. Smith, H., Nagle, C., MacVean, C., & McKenzie, C. (2016) Susceptibility of it’s possible that trophic movement of water-soluble Bemisia tabaci MEAM1 (Hemiptera: Aleyrodidae) to imidacloprid, toxins, as observed in our study, play a part in this thiamethoxam, dinotefuran and flupyradifurone in south Florida. Insects, 7, 57. global problem. Stoner, K.A. & Eitzer, B.D. (2012) Movement of soil-applied imidacloprid and In the future, exploring natural populations of thiamethoxam into nectar and pollen of squash (Cucurbita pepo). PloS One, 7, e39114. Psyllobora species located near fields that had been Sutherland, A.M., Gubler, W.D., & Parrella, M.P. (2010) Effects of fungicides on a treated with imidacloprid might help to clarify the mycophagous coccinellid may represent integration failure in disease management. Biological Control, 54, 292-299. ecological role of imidacloprid in this tri-trophic Sutherland, A.M. & Parrella, M.P. (2006) Quantification of powdery mildew removal by the mycophagous beetle Psyllobora vigintimaculata interaction. (Coleoptera: Coccinellidae). IOBC WPRS BULLETIN, 29, 281. Sutherland, A.M. & Parrella, M.P. (2009) Mycophagy in Coccinellidae: Review and synthesis. Biological Control, 51, 284-293. Supporting Information Tappert, L., Pokorny, T., Hofferberth, J., & Ruther, J. (2017) Sublethal doses of Additional Supporting Information may be found in the imidacloprid disrupt sexual communication and host finding in a parasitoid wasp. Scientific Reports, 7, 42756. online version of this article under the DOI reference: Therneau, T.M. (2015) A Package for Survival Analysis in S. In R package version xxxxx 2.38 (2015). File S1. Online supporting material.