Afidopyropen, a novel insecticide originating from microbial secondary extracts

Afidopyropen, a novel insecticide, is a derivative of pyripyropene A, which is produced by the filamentous fungus Penicillium coprobium. Afidopyropen has strong insecticidal activity against aphids and is currently used as a control agent of sucking pests worldwide. In this study, we summarized the biological properties and field efficacies of its derivatives against agricultural pests using official field trials conducted in Japan. Afidopyropen showed good residual efficacies against a variety of aphids, whiteflies and other sucking pests under field conditions. Furthermore, toxicological studies revealed its safety profiles against nontarget organisms, such as the honeybee, natural enemies and other beneficial insects, as well as mammals. Thus, afidopyropen is a next-generation agrochemical for crop protection that has a low environmental impact.


Results
Insecticidal spectra. In our study, afidopyropen showed excellent insecticidal activities against common aphid species, such as green peach (Myzus persicae), cotton (Aphis gossypii) and bean (Aphis craccivora), that damage a variety of vegetables, fruit trees, tea trees and ornamentals by sucking sap from sprouts and leaves. Furthermore, afidopyropen showed good activities against whiteflies (Trialeurodes vaporariorum and Bemisia tabaci Biotype Q), mealybugs (Pseudococcus comstocki), leafhoppers (Empoasca onukii) and psyllids, a hemipteran insect, and it exhibited good efficacies against these insect pests in field trials, while decreasing crop damage. These pests are common on many crops, such as cotton, beans and vegetables, and some pests have developed resistance to existing insecticides. Afidopyropen demonstrated good to excellent efficacy against multiple life stage of T. vaporariorum, B. tabaci Biotype Q and E. onukii. An ovicidal efficacy was not observed, but afidopyropen showed good activities against T. vaporariorum and B. tabaci Biotype Q after they hatched. It did not show insecticidal activities against Lepidoptera (Plutella xylostella), Thysanoptera (Frankliniella occidentalis), Diptera (Liriomyza trifolii), Coleoptera (Oulema oryzae) and Acari (Tetranychus urticae), indicating selectivity against  Insecticidal activity against resistant insect pests. Aphids populations resistant to commercial insecticides are appearing worldwide. Some aphids are developing resistance to OP or neonicotinoids, which are widely used to control sucking pests. Therefore, we tested the efficacy of afidopyropen against resistant populations of cotton aphids collected in Japan. Afidopyropen showed excellent efficacy against both OP and neonicotinoid resistant cotton aphids collected in fields that were equivalent or superior to its efficacy against a susceptible population (Table 2). These finding demonstrate a lack of cross resistance to OP and neonicotinoids. In addition to its excellent insecticidal efficacy against resistant populations as determined by laboratory assays, afidopyropen showed good efficacy towards populations with reduced susceptibility to commercial standards in official field trials conducted by the Japan Plant Protection Association (JPPA). In the trials, afidopyropen was used as a foliar spray at lower concentrations than imidacloprid (Fig. 2). Moreover, cotton aphids were subjected to continued selection using a foliar afidopyropen spray to assess the risk of developing afidopyropen resistance (Table 3). After breeding for 10 generations, the differences between both the LC 50 and LC 90 for afidopyropen were less than twofold between the F0 and F10.

Speed of control.
Aphids and whiteflies damage many crops by vectoring viruses that cause diseases. Therefore, it is essential for insecticides to quickly prevent sucking pests from transmitting viral diseases. Consequently, we evaluated the effects of a temporary exposure to a 10 ppm foliar afidopyropen spray of cucumber    www.nature.com/scientificreports/ leaf disks on adult aphids. After a 1-h exposure, the increase in the number of aphids discontinued, and afidopyropen's efficacy was equivalent to that of the common commercial standard flonicamid (Fig. 3).

Systemicity of afidopyropen on crops.
Given that aphids prefer to infest new sprouts and young undeveloped leaves, it is a desired attribute for aphid-controlling agrochemicals to penetrate from treated leaves to untreated young leaves or to untreated parts of leaves systemically within the plants. In tests to assess its systemicity from treated to untreated leaf surfaces, afidopyropen showed a good translaminar efficacy against the green peach aphid providing moderate to excellent control (Table 4). Furthermore, the upward systemicity from a treated to an untreated cucumber leaf was demonstrated with afidopyropen exhibiting over 95% aphid control on both leaves. However, the downward systemicity from a treated to an untreated downward leaf was lower with afidopyropen exhibiting 60.3% aphid control (Table 5). Because commercial standards for aphid control are frequently used in systemic applications, such as soil drenching or nursery box application in young seedlings, the root systemicity was also evaluated in a field trial. As a soil drenching agent, 20 mg afidopyropen per seedling showed a good systemicity against cotton aphids on cucumber, but the efficacy was inferior to imidacloprid ( Fig. 4). Furthermore, the seed-treatment efficacy was investigated by dipping wheat seeds into the insecticide solution. Similar to soil drenching tests, afidopyropen exhibited good efficacy to wheat aphid when seeds were dipped into 500 ppm of afidopyropen solution for 6 h (Fig. 5). Although the systemicity from roots and seeds were moderate in field trials (data not shown), afidopyropen possessed good systemicity in crops and controlled aphids through some exposure routes. Globally, low water volumes are commonly used when spraying pesticides. To confirm its efficacy under such conditions, we evaluated the effects of spraying at a low volume (400 L/ha) of 320 mg/L afidopyropen and at a   www.nature.com/scientificreports/ normal volume (1280 L/ha) of 100 mg/L afidopyropen. The efficacy was the same under both conditions, and the spray volume did not affect the efficacy (Fig. 6a), which was slightly better than that of flonicamid (Fig. 6b).

Management of viral spread.
Through the JPPA, we conducted field trials of an afidopyropen formulation containing 10% technical grade of active ingredient against sucking pests, and it showed good residual efficacies against aphids on vegetables and fruit trees for more than 2 weeks after a foliar application. In soy- www.nature.com/scientificreports/ bean field trials, we confirmed that 4 times 50 ppm afidopyropen foliar spray not only decreased the numbers of greenhouse potato aphids on the soybeans 6, 14 and 20 days after 1st application (Fig. 7), it also inhibited the incidence of the dwarfing disease caused by the aphid vector 62 d after 1st application. Thus, afidopyropen (50 ppm) significantly controlled soybean dwarf virus (SbDV) transmission by 27% in infested plants compared with untreated plants, as well as decreasing the numbers of aphids.

Mode of action.
Afidopyropen does not act on the nicotinic acetylcholine, GABA, glutamate, octopamine and serotonin receptors, nor on sodium channels. Furthermore, the inhibition of acetylcholine esterase, the mitochondrial electron transfer system and insect growth regulator is also not observed 19 . A recent study revealed that afidopyropen modulates the transient receptor potential vanilloid (TRPV) channels in insect chordotonal organs 20 . Chordotonal organs, which are unique to insects and crustaceans, are mechano-sensors, which are located in the joints of body segments and provide information about relative rotation of body parts. Hyperactivation and eventual silencing of TRPV channels by afidopyropen perturbs function of chordotonal organs and leads to loss of coordination, which, in turn results in inability to feed, desiccation and eventual death. Afidopyropen is classified into the 9D group of pyropene chemistry on insecticide classification by the Insecticide Resistance Action Committee 21 .
Regarding crop safety, 10% afidopyropen in water-dispersible granules has been used in a variety of Japanese field trials to confirm the insecticidal efficacy of this formulation. At an effective dose rate of 50 ppm, the foliar spray did not show any toxic effects against cereals, vegetables, tea trees, fruit trees or ornamentals.
The toxicity levels of afidopyropen against aquatic invertebrates (Table 6), honeybees (Table 7) and a variety of beneficial insects (Table 8) were low 22 . In the USA and EU, issues relating to the bee toxicity levels of existing agrochemicals are important, but a bee study using laboratory assays and semi-field trials revealed that   www.nature.com/scientificreports/ afidopyropen has limited toxic effects. The mammalian toxicity studies revealed no serious acute, sub-acute or other toxicity issues (Table 9).

Discussion
In this study, we revealed that afidopyropen had excellent insecticidal activity against devastating piercing and sucking agricultural pests, although its insecticidal spectrum was narrow. The narrow spectrum might result from the modes of action of the TRPV modulators that help insects recognize outside mechanical stimuli and maintain proper posture and behavior in response to the stimuli, and we observed only weak activity with Cpd. 1 on Plutella xylostella lepidopteran larvae. The efficacy differs among the insect stages. Since hemipteran larvae are aggressively seeking foliage for feeding, the efficacy is especially high. A behavioral abnormality has also been observed in some adult hemipteran insect species, and it halts the population's growth. Hemipteran pests damage almost all crops, genetically modified and non-genetically modified, worldwide, and even with existing insecticides, they are still not sufficiently controlled. Drug resistance and/or registration issues owing to toxicological and eco-toxicological properties of existing insecticides have negatively impacted control strategies. In particular, those of the main target pest, the aphid, with its short lifecycle and ability to quickly develop insecticide resistance. In fact, the development of resistance against major insecticides, like neonicotinoids, is remarkable 23,24 . Afidopyropen has a novel chemical scaffold and does not show cross resistance with major insecticides such as OPs, synthetic pyrethroids and neonicotinoids. In some target insects, like whitefly, even pymetrozine, which acts on TRPV, the same target protein as afidopyropen, have resistance issues develop owing to metabolic factors, but afidopyropen shows a good efficacy against pymetrozine-resistant whiteflies 25 . Moreover, our selection study using a foliar afidopyropen spray showed a low risk of resistance development. Afidopyropen is an insecticide effective by foliar spray, not only in high volume, but also in low volume applications which are globally used practices. Afidopyropen has good translaminar efficacy and systemic activity and has suitable systemic properties to control sucking pests that prefer new shoots and seedlings. In addition, afidopyropen has good control efficacies against aphids when used in soil drench and seed dip applications, as well as a good control efficacy against whitefly when used in soil drench (data not shown). However, the dose rate is higher than those of standards. Further investigations would be needed to find suitable useful scenarios in systemic uses. Although the speed of  Table 9. Mammalian toxicity studies using afidopyropen.

Study Effect
Acute oral toxicity to rat LD 50 > 2000 mg/kg www.nature.com/scientificreports/ kill was slow, insects treated with afidopyropen showed abnormal behaviors in a few hours and became unable to damage plants in a short time. In fact, in some field trials, treatments stopped the development of diseases resulting from aphid-vectored viruses and very few remaining dead insects were observed because they easily fell off the treated leaves. As documented in Japanese official field trials on a variety of crops, afidopyropen has shown excellent efficacy for the control of aphids, whiteflies, leafhoppers and mealybugs. As well, regarding consistency of efficacy across the pest spectrum overseas, afidopyropen exhibits excellent field efficacy against aphid, whitefly and the Asian citrus psyllid at low doses, 10-50 gai/ha, by BASF [26][27][28][29][30] , and the Food Safety Commission of Japan information indicates that it is not persistent in the environment 22 . For instance, the DT 50 for afidopyropen is 2.7-18.6 days in soils under aerobic conditions, and 1-2 months under simulated sunlight. After application to crops, it is relatively labile with low persistence. The main metabolite, its dimer, has low acute and sub-acute toxicity levels as well.
In addition, afidopyropen shows low toxicity levels against honeybees and natural enemies, as well as against mammals. Because of environmental dynamics, afidopyropen is expected to be an eco-friendly tool of sustainable agriculture. Now that afidopyropen has been launched by BASF SE and entered into the crop protection market worldwide, it will aid in achieving sustainable agriculture. Moreover, we seek opportunities to extend this technology into unexplored crop production segments and application scenarios to improve current practices and contribute to enhanced crop productivity, including systemic uses and the treatment to control other pests and synergistic combinations.

Methods
General. Afidopyropen was produced and purified in accordance with our established methods 31 . The commercial insecticides imidacloprid and flonicamid were purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Organic solvents, chemical reagents and all the consumables were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
The authors confirm that we used plants commercially available in Japan in this research and all studies involving plants and plant materials were done in compliance with local and national regulations/guidelines.

Insecticidal assays. Laboratory insecticidal tests against agricultural pests. Afidopyropen was evaluated
using each target insect pest on leaf disks or potted plants in accordance with our previous report 32 . The test conditions are summarized in the Supplementary Information section. Resistant populations and populations present on 0.002 ppm afidopyropen (50% acetone/distilled water containing 0.05% Tween 20) solution-treated cucumber leaves were also tested with cotton aphids using the same leaf disk assays. Cabbage (Kinkei 201, purchased from Sakata), cucumber (Suyo, purchased from Sakata), fava bean (Funaokaissun, purchased from Takayama seed), kidney bean (Celina, purchased from Takii), wheat (Nourin 61, seeds collected inhouse), rice (Jikkoku, seeds collected inhouse) or tea (Yabukita planted inhouse) plants were used for each test, which are available in Japan. LC 90 was calculated in a probit method with ECOTOX v3 software.
Speed of control assay. Adult cotton aphids were exposed to glass dishes treated with each insecticide after drying for 1 h. Then, five treated adults were placed on a cucumber (Suyo) leaf disks. At 1, 48, 120 and 168 h after infestation, the numbers of live adults and larvae were counted. The test was conducted with two replicates.
Translaminar assay. At 7 days after treating only the upside of an eggplant (Senryou 2, purchased from Takii) true leaf with each afidopyropen solution concentration, four adults of green peach aphids were placed on the opposite untreated leaves of the 2.8 cm diameter of leaves cut from the treated eggplants. The number of aphids on the opposite untreated sides was counted. The test was conducted with three replicates.
Systemic activity from treated leaves. At 4 days after treating only the 1st or 2nd cucumber (Suyo) true leaf with 1000-ppm afidopyropen, three adult cotton aphids were placed on the leaves. The numbers of aphids on both the treated and untreated upward/downward leaves were counted after 8 days. The test was conducted with two replicates.
Soil drenching efficacy. When transplanting 3-week-old cucumber (Suyo) seedlings, afidopyropen formulation or a commercial insecticide imidacloprid was applied into each transplantation hole. At 7, 14, 21, 28 and 35 days after transplantation, the numbers of aphids on 72 leaves from 12 plants were counted. The test was conducted in triplicate, with 12 plants per replicate.
Seed dipping efficacy. The wheat (Nourin 61) seeds were dipped in a water-based dilution of 5% wettable afidopyropen powder for 6 h. After the treatment, seeds were allowed to put up shoots for 72 h. Then, the seeds were transplanted. At 2 days after transplantation, 10 adult wheat aphids were placed onto the seedlings. At 1, 4, 6 and 8 days after infestation, the number of aphids was counted in each plot. This test was conducted in triplicate.
Efficacy of foliar applications against cotton aphid (A. gossypii) on potato. A JPPA field trial was conducted in 2011 using 5-week-old potato (Nishiyutaka, available in Japan) plants. A solution of afidopyropen dissolved in water was applied at 2000 L/ha to potatoes naturally infested with the cotton aphid. At 3, 7 and 14 days after application, the number of aphids was counted on 10 plants in each plot. Each plot was tested in triplicate. Management of viral spread. A field trial was conducted in 2010 by the Hokkaido Plant Protection Association using approximately 2-month-old soybean (Toyomusume, available in Japan) plants. A solution of afidopyropen dissolved in water was applied at 150 L/ha to soybeans naturally infested with the glasshouse potato aphid Aulacorthum solani. At 3, 7 and 13 days after application, the number of aphids was counted on 10 plants in each plot. Furthermore, the number of plants infested by SbDV was counted 13 days after application. Each test was conducted in triplicate. The control rate (%) for infested aphids was calculated using Eq. (2), and SbDV infection (%) and control rate (%), compared with untreated plants, was calculated using the following formulae: