CORDIS - EU research results

Ecofriendly synergists for insecticide formulations

Final Report Summary - ECOSYN (Ecofriendly synergists for insecticide formulations)

Executive Summary:
The world-wide use of synthetic insecticides to control insect pests has led to insecticide resistance and to environmental contamination. One possible route to reduce insecticide use without compromising control is to use a synergist in combination with insecticide. Synergists are themselves nontoxic but act by increasing the effectiveness of the insecticides they are used with. The mode of action of most synergists including the well-known compound piperonyl butoxide (PBO) is to inhibit the metabolic systems in insects that metabolise or sequester insecticide molecules. As a result they can increase the sensitivity of insecticide-susceptible insects and also delay and/or overcome metabolic resistance, a common form of resistance mediated by enhanced production of detoxifying enzymes.
The EcoSyn project consists of a consortium of experts to optimise the use of synergists in agriculture by elucidating the relationship between synergist chemical structure and activity on a variety of target insect metabolic enzymes and efficacy to resistant and susceptible pest insects and beneficials in a laboratory and field environment. The EcoSyn project aimed to identify and develop synergists for insecticide formulations in both agricultural and public health applications, enabling deployment strategies that enhance the effectiveness of several important classes of insecticide.
An important aim was also to enable a reduction in the amount of insecticidal active applied whilst maintaining efficacy, thereby reducing any adverse effects of these insecticides on beneficial insects such as bees. As such, the novel synergists may be defined as eco-friendly.
Due to complex and multidisciplinary efforts required in the project, the work was structured into nine work packages to cover major scientific challenges, development and validation and dissemination as well as IP exploitation.
Scientific and innovation aspects were addressed for production of esterases and recombinant P450 interactions with enzymes, modelling, synthesis and laboratory bioassays of novel synergists, glasshouse and semi-field tests and potential of piperonyl butoxide /novel synergists to select for resistance. In particular this last activity has concerned the long-term stewardship of piperonyl butoxide giving the scientific basis for the further studies of the novel synergist developed during the project. These studies were not carried out within the project due to its short time but will be matter of novel studies being a preparatory objective for registration of possible novel synergist considered for commercialization.
The studies started with the interaction detection of the potential novel synergists on Mixed Function Oxidases and esterases of selected insects such as Myzus persicae and Bemisia tabaci , being these insects taken as representative for agro pests. In combination with the experimental results and modelling studies that gave indications for the best suitable chemical structures, many synergists were synthesized . Through a such iterative process, a selection of the possible synergists was made, based on the best interaction with the previous mentioned enzymes and the selected novel synergists were tested in the lab on Myzus persicae, Bemisia tabaci and Meligethes aeneus as representative for agro pests and on Musca domestica and Blattella germanica as representative for no agro pest. The potential novel synergists were tested in combination with commercial pyrethroids, neonicotinoids and an insect grow regulator (methoprene). A further selection was made also by testing the selected synergists in glasshouse and semi field conditions, bringing to a selection of two novel synergists with good activity and potentially exploitable viz. EN1-218 particularly suitable for agro application and EN1-216 for public health. A particular attention was put to the costs of the selected synergists taking into account the cost/performance ratio and the commercial piperonyl butoxide as a comparison . It’s worth mentioning that both synergists enable a reduction of up to ½ the field rate dose of the insecticides and can therefore be termed eco-friendly.
Dissemination activities were performed through papers, workshops and communications.
A special attention was put to the IP protection and, in agreement with the participants and the rules of the signed Consortium Agreement three novel patent applications of novel synergists were filed on September 2015: two European Patent Applications and one Italian Patent Application.

Project Context and Objectives:
Insect pests represent a threat to agricultural production and the health of humans and domestic animals through crop damage and vector transmission of several important diseases. The economic impact of insect pests is huge in terms of human and animal health - as well as for agricultural production.
Chemical insecticides have been, and remain, the mainstay for controlling insect pests and represent a guarantee for the supply of affordable food as well as a tool for an effective disease vector control for the foreseeable future. Unfortunately, the world-wide use of high levels of synthetic insecticides to control insect pests over many years has led to increased selection for insecticide resistance and has contributed to environmental contamination.
Resistance leads to wasteful and ineffective insecticide treatments and may prompt a return to chemicals with less favourable environmental profiles as used in the past. Central to these issues is the emerging problem of how to meet the challenges of a growing population by producing more food whilst minimising negative environmental impacts (sustainable intensification). For both crop protection and vector control this needs to be achieved without a simultaneous escalation in insecticide usage.
The World Health Organisation (WHO) defines resistance as “the inherited ability of a strain or organism to survive doses of a toxicant that would kill the majority of individuals in a normal population of the same species”
Resistance to insecticides most commonly evolves through two main mechanisms;
1)enhanced production of metabolic enzymes that break down or sequester the insecticide before it reaches the target protein (termed metabolic resistance)
2)mutation of the insecticide target protein resulting in a loss or reduction in insecticide binding (termed target-site resistance). These mechanisms may exist individually in an insect or may be found in combination where the overall resistance they confer is substantially greater.
Three main classes of enzymes have been shown to be involved in the detoxification of insecticides and metabolic resistance. These are esterases, cytochrome P450s (P450s) and glutathione-S-transferases (GSTs)
Esterases and P450s are involved in phase 1 detoxification as they can act on the intact insecticide, whereas GSTs are phase 2 enzymes, acting on degradation products of phase 1 enzyme activity. Synergists can increase the efficacy of insecticides by inhibiting one or more of these metabolic enzyme families. Because both resistant and susceptible insects express these enzymes, inhibition can make susceptible populations hyper-sensitive to insecticides in addition to overcoming the resistance of insects that over express these enzymes.
PBO (piperonyl butoxide) is a well-known synergist and is widely used in the household market. As yet, it has only a niche market in agriculture. PBO is now commonly applied with pyrethrins, synthetic pyrethroids and many other insecticidal actives. Many studies in the literature report on the mechanisms of action of PBO, initially as an inhibitor of cytochrome P450s and subsequently of resistance-associated esterases. Currently, progress has been made regarding the interaction of PBO with a resistance-associated esterase (E4) and with the major human detoxifying P450, CYP3A4. However, such studies have not been carried out on recombinant enzymes known to be responsible for conferring insecticide resistance. Furthermore, there have been no studies to investigate the effect of selection by the use of a synergist in combination with a decreased insecticide dosage compared with selection of insecticide at the field rate.

In terms of advancing knowledge and technical progress, the project is aimed to clarify the interaction of PBO (as the standard product) with the insect defence enzymes. On the basis of these interactions, to define and model potentially more potent structures, with a view to implement eco-friendly synergists for use with insecticides in both agriculture and Public Health applications through the following steps:
•Elucidation of the mechanism of action of the standard synergist, PBO, widely in current use in household and PCO applications.
•Design of novel synergists based on experimental results, SAR of PBO and existing analogues, molecular modelling whilst maintaining important physico-chemical parameters.
•Choice of appropriate agriculturally-important susceptible and resistant pest insects
•Examples of beneficial insects found in the same environment.
•Choice of appropriate susceptible and resistant disease vector insects (Public Health).
•Choice of appropriate insecticide actives for evaluating synergistic effects on susceptible and resistant populations of economically important insect pests.
•Evaluation of the activity on pest and beneficial species using laboratory bioassays and field trials.
•Evaluation of the potential of synergist/insecticide application to select for resistance compared with application of insecticide alone.
•Process development to obtain the definitive synergist(s) with the aim of achieving an industrial and economically viable process.
•Definition of global patent rights to protect the invention.

A further objective of the project was the promotion of the novel synergists in combination with reduced levels of insecticidal actives as part of an integrated pest management strategy.
As the aim of the project involved selection of possible synergists for commercial opportunity, consideration was based on the cost performance ratio, as well as synthesis pathways.
The project was structured on this basis in seven different work packages (WP) as follows:
- Elucidation of the mechanism of action of the standard synergist PBO and existing analogues using purified or recombinant P450 and subsequent interactions through a structure-activity relationship (SAR) between the purified enzymes and piperonyl butoxide and existing analogues ( WP1 and WP2)
- Design of novel synergists based on experimental results, SAR, molecular modelling and their synthesis. As one aim of the project was consideration of commercial opportunity, a preliminary cost estimation was taken into consideration (WP3)
- Evaluation of the efficacy of the novel synergists on pest and beneficial species using laboratory bioassays (WP4)
The evaluation was carried out on agriculturally important pests such as Myzus persicae (M. persicae) (green peach potato aphid), Bemisia tabaci (B. tabaci) (cotton whitefly), Meligethes aeneus (M. aeneus) (pollen beetle) and on beneficial insects such as Apis mellifera (A. mellifera) and Bombus terrestris (B. terrestris).
For vector disease (public health) insects, Blattella germanica (B. germanica) (German cockroach) and Musca domestica (M. domestica) (common housefly) were used.
To evaluate synergistic effects on susceptible and resistant populations of economically important insect pests, different classes of insecticide with differing modes of action and application were considered.
-Cypermethrin a pyrethroid
-Tau fluvalinate a pyrethroid
-S-Methoprene an IGR
-Imidacloprid a neonicotinoid
-Thiacloprid a neonicotinoid
Due to restriction of neonicotinoid application in some countries such as Turkey, thiamethoxam was also included

- Evaluation of the activity of the novel synergists on pest and beneficial species using glasshouse and semi-field trials (WP5)
The evaluation was carried out on the basis of a chosen range of the novel synergists in semi-field conditions and by using the same insecticides as mentioned above.

- Evaluation of the potential of synergist/insecticide applications to select for resistance (WP6)
Field populations of B.tabaci and M. persicae were collected and bioassayed with a pyrethroid and a neonicotinoid in combination with PBO, providing selected and unselected strains for molecular characterisation. Further characterisation of the change in gene expression between the selected and unselected pools and between those selected with insecticide alone or insecticide+PBO can be used to evaluate whether a regime incorporating a synergist increases selection for single, or multiple, resistance genes.

-Validation and Process development to obtain the definitive synergist(s) (WP7)
The novel synergists identified on the basis of the tests carried out in WP4 and in WP5 were validated. Furthermore, a development study was carried out to confirm the chemical feasibility from an industrial point of view, taking into consideration the costs of the intermediates as well as the safety of different chemical steps.
Another important point for consideration was the patentability of the novel synergist(s).
This was agreed by all SMEs, in accordance with the Consortium Agreement signed among the participants.
Two other work packages were utilised; Dissemination of the results (WP8) and the Project management (WP9)

Project Results:
The project was divided in nine work packages which are described below, together with results obtained.

The project began with the isolation and purification of specific metabolic enzymes (esterases and P450s) that have been reported to be responsible for conferring resistance to insecticides in economically important crop pests. The esterases were prepared from a bulk collection of insects via purification, whilst the P450s were prepared recombinantly (CYP6CM1 and CYP6CY3). For the latter, reduced CO-difference spectrum revealed a classic Soret peak for both CYP6CY3 and CYP6CM1 at 450 nm indicative of a stable, good quality functional enzyme. In initial enzyme assays of P450 expressed using Sf9 cells activity was demonstrated for both P450s although greater O-dealkylation activity of two fluorescent model substrates MFC (7-Methoxy-4-(tri-fluoromethyl) coumarin) and EFC (7-ethoxy-4-trifluoro-methylcoumarin) was revealed for CYP6CY3 than CYP6CM1. When the expression of CYP6CM1/CYP6CY3 was repeated using the Tn5 cell line from Trichoplusia ni (Lepidoptera: Noctuidae) the specific activity of the microsomal preparations was significantly enhanced. Therefore these preparations were selected for use in the subsequent WP2 with the aim to assess the activity of the novel synergists. Purification of esterase was successful, with enzyme assays using 1-naphthyl acetate as substrate confirming high quality functional esterase had been obtained.

The aim of WP2 was to assess the inhibitory potency of the novel synergists against P450s and esterase(s) from WP1. Inhibition of the insect defense enzymes was achieved by monitoring the decrease in activity against model substrates (7-ethoxycoumarin (7-EC) for CYP6CM1 and 7-ethoxy-4-trifluoromethylcoumarin (EFC) for CYP6CY3; 4-nitrophenyl acetate (pNA) for FE4 and 4-nitrophenyl octanoate (pNO) for B. tabaci esterases). Each enzyme / inhibition SAR (Structure Activity Relationship) delivered its own bespoke predictions for increased potency as tabulated in Deliverable 2.1. Thus, no single structure could be predicted as the ‘ultimate’ structure to confer highest inhibition against all enzymes, but rather it would depend upon the nature of the resistance conferred (i.e. predominately P450-based etc). However, the SAR supplied evidence that alkynyl structures with particular alkyl chain length would be the basic structural template to confer high inhibition.
Table 1 “Remaining activities for CYP6CM1, CYP6CY3 and FE4” shows the sum of activity remaining for CYP6CM1, CYP6CY3 and FE4 after treatment by the potential novel synergists in comparison with the commercial synergist piperonyl butoxide. Enzyme activity remaining (%) CYP6CM1 + enzyme remaining (%) CYP6CY3+ enzyme activity remaining (%) FE4 = total activity remaining given in Table 1. If no inhibition occurred, the total would be 300.
The first 4 structures are the same basic template, predicted by WP2 to confer highest inhibition (and thus synergism). Protein modelling in silico was completed using closest templates from the PDB database and Autodock vina. No assumptions were made about the docking location of the ligand, the grid box encompassing the whole protein. It was found that docking locations were in agreement with locales reported to be responsible for metabolism of insecticides from the literature i.e. serine 387 and arginine 224 in B. tabaci. Binding affinities (free energy) of the potential novel synergists were in agreement with the inhibition potency.

The aim of WP3 was to synthesize the potential novel synergists on the basis of the Structure Activity Relationship (SAR) evaluation and in- silico modelling. The activities were carried out in the laboratory at the gram scale in close cooperation with the leader of WP2. In this way the work was developed as an interactive effort to identify the optimal products based on in vitro analysis. At the same time a literature search was carried out to confirm the originality and chemical feasibility of the potential novel synergists.
The potential novel synergists had structures basically attributable to three chemical families: benzodioxole derivatives containing a triple bond, benzodioxole derivatives containing an alkoxyalkyl chain and 2,3-dihydrobenzofurane derivatives. The literature search suggested that most of the products were novel and potentially patentable. All of the structures were checked using two different techniques, viz MS and 1H and 13C NMR. Particular attention was given to 2, 3-dihydrobenzofurane derivatives where the definition of positional isomers was required. In this instance, the analyses were carried out through gCOSY NMR and verified, where possible, through X- rays. All the products were checked for their activity in vitro as reported in WP2 and particular attention was given to the synthetic and economic feasibility of the products as potential novel synergists, taking into account the cost/performance ratio to be assessed in vitro and subsequently validated in vivo. A main objective of the project was to identify potential novel synergists to be used in agro applications, and the chemical process cost evaluation was a key factor for their -potential implementation.

WP4 used laboratory bioassays to assess in vivo efficacy of pyrethroids (cypermethrin/tau-fluvalinate), neonicotinoids (imidacloprid/thiacloprid) or IGR (S-Methoprene), alone or in combination with PBO (piperonyl butoxide ) / novel synergists, on different populations of important agricultural and household pests (M. persicae, B. tabaci, M. aeneus, B. germanica and M. domestica) possessing various levels of resistance to insecticides. Simultaneously, laboratory bioassays evaluated the effects of the above products on honeybees / bumblebees. One susceptible (1X) and two resistant clones (96H and 92H6) of M. persicae (aphids) were used in bioassays: the latter originated from individual parthenogenetic females from field populations collected in Italian peach orchards in 2010 (92H6) and 2011 (96H) after neonicotinoid and pyrethroid application failures. A highly resistant population of B. tabaci (whitefly) was collected from ornamental plants in glasshouses in Northern Italy after control failures with several insecticide classes. Meligethes aeneus (pollen beetle) populations were collected in Northern Italy and Poland. In assays using M. domestica (house fly) two internal standard populations with different levels of insecticide susceptibility were included.
All bioassays used commercially available formulations of alpha-cypermethrin (Fastac), tau-fluvalinate (Mavrik 20 EW), imidacloprid (Confidor 200 SL), thiacloprid (Calypso), cypermethrin (Cymina) and S-methoprene (Biopren 20EC). Bioassay methodology reflected the combination of pest, insecticide, synergist and endpoint. A baseline for each combination of pest and insecticide was determined and a diagnostic dose conferring 25-35% mortality was used in subsequent tests to identify synergism levels of the novel synergists. Preliminary bioassays were conducted using both technical grade and EC formulated synergists (PBO and EN1-126 (EN126) with split applications. Subsequently, EC formulated synergists only were used in tank-mix application against resistant clones. The EC formulation alone was not toxic. Mortality data (percentage arcsin transformed) were statistically analysed using analysis of variance (ANOVA). Significant differences were observed in neonicotinoid treatments with clone 96H. All the Calypso + synergist mixtures (except EN1-219) produced significantly higher mortality than Calypso alone.
M. persicae
Occasional high mortality produced by EN1-213 alone was observed in clone 96H, whilst in clone 92H6 high mortality was produced by EN1-218 and EN1-126. Synergistic ratios (Insecticide + synergist efficacy / Insecticide alone efficacy) recorded in these tests are reported in Table 2. “M. persicae. Synergistic ratios calculated for a discriminating dose of the selected insecticides and EC formulated novel synergists, applied at 1 g /L, without split application (tank mix), against two resistant clones of M. persicae. Darker cells represent higher synergistic ratio values.”
In bioassays using Confidor, only the mix with EN1-126 was statistically different from Confidor alone. No significant differences were observed with Fastac. In tests with Mavrik significant differences were present only between mixtures of the insecticide plus synergists and synergists alone. Mixtures were not different from insecticide alone.
In bioassays with clone 92H6 and neonicotinoid mixtures, insecticide plus synergist were significantly different from insecticide and synergist alone. No significant differences were observed for Fastac. With Mavrik, EN1-215 was the most effective synergist.
Synergistic ratios (Insecticide + synergist efficacy / Insecticide alone efficacy) calculated from tests against B. tabaci are reported in Table 3 B. tabaci. Synergistic ratios calculated for a discriminating dose of the selected insecticides and technical grade novel synergists in acetone, applied at 0.5 /L, 5 hours before insecticide application, against a resistant population of B. tabaci. Darker cells represent higher synergistic ratio values.
Mortality data (percentage arcsin transformed) were statistically analysed using analysis of variance (ANOVA). Mortality observed in whiteflies treated with synergists alone was low and not statistically different from that observed in the untreated group. Highest mortality was observed in those groups treated with insecticide+synergist, but this was not always significantly different from insecticide alone. As with M. persicae it was decided to adopt a tank mix bioassay using EC formulated synergists. Again, the number of synergists to be assayed was restricted according to results from the in vitro assays.
The synergists were used at two different rates: 0.5 g/L and 1 g/L. The products were applied using a Potter spray tower. An increase in efficacy as well as in synergistic ratios was observed when the synergist application rate was increased.Baselines of synergist only toxicity against peach-potato aphids were estimated using one susceptible (1X) and one resistant (92H6) clone. Results are shown below. When applied against the susceptible clone, PBO (at concentrations 0.05 g/L to 10 g/L) gave only low toxicity). The standard concentration (1 g/L) was non-toxic for PBO, EN1-216 and EN1-218. The highest toxicity was recorded with EN1-126 and EN1-215 at 10 g/L.All synergists (at concentrations up to 5 g/L) used against the resistant clone (92H6) gave low mortality. The highest toxicity was observed for EN1-126, EN1-215 and EN1-216 at 10 g/L.
As no laboratory rearing protocol exists for Meligethes aeneus species, field collected populations were used in vial bioassays. Populations were collected from Northern Italy and Poland in spring 2014 and 2015. The only novel synergist tested in 2014 was EN1-215, at concentrations of 0.1 and 0.01 g/L., PBO and EN1-126 were also tested, for comparison. Synergists alone, at a concentration of 0.1 g/L, were toxic and so only the 0.01 g/L dose was used. Considering the three Polish populations collected in 2014, PBO synergised pyrethroids in all three and neonicotinoids in population 1; whilst for populations 2 and 3 the best performance with neonicotinoids was achieved with EN1-215 . In 2015 an Italian population was tested and the highest synergistic ratios were observed with Confidor: EN1-126 was the most potent, followed by EN1-215 and PBO. Synergistic ratios were lower with Calypso, but the ranking was similar to that of Confidor. With pyrethroids the highest ratios were obtained by EN1-215/EN1-216 followed by EN1-126 when applied together with Fastac; with Mavrik the ranking was EN1-126/ PBO followed by EN1-215 . Using a Polish population, the highest synergistic ratio was observed with Confidor: PBO, EN1-213 and EN1-215 were the most potent, but EN1-126 was the least toxic when used alone. Synergistic ratios were lower with Calypso, where EN1-126 produced the highest ratio as a consequence of its lower innate toxicity. Considering Fastac and Mavrik, all the synergists were equipotent, with higher synergistic ratios observed with the latter. Blattella germanica
Bioassays with Cypermethrin
Following the estimation of a dose-response for Cymina (cypermethrin) against B. germanica a discriminating dose of 120 µg (a.i.)/mL was applied together with 1 mg (a.i.)/mL of synergist.
Knock down was monitored every 5 minutes up to 30 minutes and mortality 24 hours later. Only EN1-213 produced an increase in knock down in comparison with the insecticide alone. Knock down produced by synergist alone was usually negligible (Figure 4). No toxic effects were observed with synergists alone: only EN1-213 produced a very low mortality. All the synergists mixed with Cymina produced a significant increase in mortality 24 hours after the application in comparison with the synergists and the insecticide alone. Mortality produced by the synergists alone was never statistically different from the mortality of the untreated control. Bioassays with S-methoprene (Insect Grow Regulator- IGR)
S-methoprene is an active substance that mimics juvenile hormone, essential in the development of insects. When IGR formulations are tested, the development of the tested species and their offspring is monitored rather than mortality. The mechanism of action of an IGR is to arrest development of insects and their colonies. Thus, IGR trials have a long end-point, with mortality resulting from deformation and suffocation at the pupae stage.
To test the efficacy of S-methoprene against adults, a novel bioassay procedure was developed: single virgin females were isolated and fed for 7 days with approximately 1 mL of an artificial diet obtained by mixing 20% fish feed (powder), 0.25% S-methoprene (a.i.) and synergist used at 1:3 ratio and water to 100%. After 7 days females were removed and mixed with males for mating and checked daily for egg case development, abortion, hatching of larvae and female mortality. Mortality of females was quite low and no mortality was observed in untreated control specimens. There was a significant reduction of offspring from treated females but this effect seemed to be transient and linked to the general reduction of fertile egg case production rather than a reduction of the vitality and number of eggs developing inside the ootheca produced by the treated female.
Egg case production was reduced by applying EN1-215 + methoprene and EN1-216 + methoprene. All the treatments induced abortion in the range of 40 to 80 % of the produced oothecae with the exception of EN1-216. The second egg case aborted from 20 to 100%. Often only 50% of egg cases produced viable larvae.
A statistically significant reduction in the total number of emerging larvae was observed with the first egg case. A delay in the production of the first egg case was observed compared to the untreated control, but not in the time between egg case production and hatching, nor the production and hatching of the second egg case.
Tests with imidacloprid were abandoned, as agreed with the Co-ordinator and the Project Officer, due to the inability to develop / formulate a gel with imidacloprid and potential novel synergists.
Musca domestica
Cypermethrin EC formulation and an Insect Growth Regulator, S-methoprene EC formulations (with, or without PBO and novel synergists) were studied and evaluated.
Test samples were supplied by UCSC and Babolna Bio. At the start of the studies, baseline toxicities were determined, and in both cases very low doses chosen.
Standard surface (glass) trials were chosen for cypermethrin studies, initially with susceptible and semi-resistant “A” and “B” populations, followed with the WHO strain. For IGR larvae studies, a special manure was selected in which the development (and/or the emergence inhibition) of M. domestica larvae were observed.
Following a number of trials, EN1-216, EN1-218 and EN1-126 were identified for further evaluation with a very low dose of Biopren (2.5 mL). Results showed equipotent synergism, all superseding Biopren 20 EC alone.
Beneficial insects
A study of possible adverse effects of the synergist/pesticide regime on beneficial insects was conducted. Bioassays with pesticides and the novel synergists were performed on Apis mellifera (honeybee) and Bombus terrestris (bumblebee). A preliminary evaluation on synergist toxicity was carried out using acute oral and contact assays including PBO for comparison. Overall, toxicity for the synergists alone was low except for EN 1-215 which was eliminated from further studies.
All the synergist bioassays were performed with EC formulations, as applicable for field conditions. Two pyrethroids (alpha-cypemethrin-FASTAC and tau-flavulinate-MAVRIK) were tested in combination with the synergists as well as two neonicotinoids: thiacloprid (CALYPSO) and imidacloprid (CONFIDOR). With the exception of imidacloprid, no synergism was observed.

The aim of WP5 was to evaluate the novel synergists chosen in WP4 (laboratory tests) in glasshouse and in semi-field conditions, to enable an informed decision based on conditions close to real applications. Based on the in vivo results obtained in the laboratory, the novel synergists used were EN1-126, EN1-213, EN1-215, EN1-216 and EN1-218. EN1-215 was included on the basis of laboratory tests on B. germanica, even though the preliminary evaluation of the toxicity tests on pollinators was undesirable.
The novel synergists were evaluated on Myzus persicae, Bemisia tabaci, Meligethes aeneus and Musca domestica.
Myzus persicae
The proposed field trials in UK with aphids on oilseed rape was not carried out due to destruction of the trail area by cabbage stem flea beetles. Replacement trials were conducted in glasshouses during the winter 2013-2014. Results of synergism studies, including PBO for comparison, on oilseed rape infested with aphids at Debanham, Suffolk, UK, were negative. However, pymetrozine ( Plenum), a feeding blocker for aphids, gave positive results. The hypothesis for the negative effect was attributed to the difficulty in hitting the target insects feeding on the undersurface of large leaves in a field situation. The importance of formulation plays a very important role in the success or failure of the trials and will need to be addressed in further developments.
Further trials on other insects were carried out in Turkey.
Results of tests conducted using Aphis gossypii on lettuce (in the absence of significant infestations of M. persicae) showed a high mortality produced by EN 1-218 in combination with thiamethoxam and cypermethrin when compared with the actives alone.
Bemisia tabaci
Preliminary trials set up in UK to test the concept of using synergists with insecticides to control pests, such as whiteflies in tomatoes, were unsuccessful. In the case of tomatoes, it was discovered during the course of raising plants for the proposed trial with whiteflies, that the glasshouse facilities to be used to carry out the trial would not/did not pass strict inspections by British government authorities for a non-indigenous licensable pest. Further work on whiteflies was therefore transferred to AITY in Turkey, where such restrictions and licenses were not required, as the pest was indigenous there.
A set of experiments against natural resistant populations of Bemisia tabaci were carried out.
Three different trials with synergist and pesticide combinations on Bemisia tabaci provided useful results. In these field (glasshouse) experiments the following synergists were used: EN1-218 (1-218), EN1-126 (1-126), EN1-215 (1-215), EN1-213 (1-213) and piperonyl butoxide (PBO), combined using split application (4 hours) or a tank-mix with the following insecticides (commercial formulations): deltamethrin, cypermethrin, thiamethoxam and imidacloprid.
The crops were: pepper, eggplant and cucumber and a randomized block design was used in the experiments
The preliminary results did not differentiate between the novel synergists. Thereafter, synergists were combined with a reduced (2/3) dose of the insecticides. The insecticides used were limited to cypermethrin and thiamethoxam.
Meligethes aeneus
Trials were set up to test the efficacy of the novel synergists in comparison with PBO in combination with Fastac (alpha-cyperrmethrin) and Plenum (pymeytrozine ) using pollen beetle on mustard, before flowering, when pollen beetle abundance was highest. .
The synergists used were EN1-126, EN1-215, EN1-213 ,EN1-218, EN1-216 and PBO as a comparison with the following treatments:-
a) Plenum 500 WG (0.15 kg/ha)
b) Fastac 100 EC (0.12 L/ha)
c) PBO 400 mL/ha
d) Novel synergists 400 mL/ha
Before treatment, the average number of pollen beetles per plant was calculated. Then the same calculation was made 1, 3, 7, and 11 days after the treatment. All these calculations were based on the numbers of pollen beetles on 100 plants within each plot.
On the basis of these calculations the efficacy of all combinations was established, according to the Henderson-Tilton’s formula
Efficacy [%] = (1 - n in Co before treatment * n in T after treatment /
n in Co after treatment * n in T before treatment) * 100
Where n = average number of insect per one plant, T = treated , Co = control
A reduction in the mean number of insects per plant was recorded particularly for 1 and 3 days after the application in all the treatments where insecticide + synergist were sprayed. The efficacy of all the products was high just after the application, but 3 DAA the insecticide + synergist mixtures maintained their efficacy. The highest synergistic ratio was observed 3 DAA.
Musca domestica
Further tests were performed using PBO and EN 1-216 adopting the same protocol as reported previously in WP4. In these tests, Cymina performance (efficacy) was poor (67.01% mortality), however this result had been achieved within almost 10 minutes and although after 40 minutes the result was 78 %, due to revival ended with 67%. It became quite clear that while the knock-down capacity is acceptable, the killing effect is not strong enough and does not reach the standard requirement of 90 % (but this may be due to be the fact that very low doses were used). Cymina + EN 1-216 EC acted extremely well (98.97 %), kept the speed of action and performed the result within 20 minutes.
The efficacy of PBO alone (6.06 g) was 87.63 % which is extremely high, but acted somewhat slowly at the beginning, with the killing effect speeding up after 90 minutes. PBO was used at two different application rates (3.03 and 1.01). 3.03 g PBO gave 71.43 %, but at the beginning of the trial PBO showed a very slow effect. 1.01 g PBO still gave 61.24 % with the same slow mode of action.
In order to find out whether any of the PBO 80 EC components had additional killing effects, Endura prepared a blank 80 EC formulation without PBO. The results showed no effect, therefore only PBO is considered responsible for the synergism.
These results clearly show that in the susceptible strain the results were excellent and have even surpassed the Cymina alone results. In the case of the less susceptible strain, the results were still good, considering the low concentration of the synergists. The overall conclusion of these studies is that the chosen synergists all have a certain killing effect and as such can be considered as an insecticide against the housefly.
A complete ANOVA test considering data from all the experiments was statistically significant. Mean mortality comparisons differentiated well between efficacy of insecticide alone and insecticide plus synergists. Efficacy of EN1-216 combined with Cymina was the highest and statistically different from all the remaining treatments. In both lines of study EN1-126, EN1-216 and EN1-218 performed above or close to PBO, but all performed well and have increased the efficacy of the insecticides.
Further studies however are necessary to establish the efficacy and mode of action in the case of resistant public health insects.
Beneficial insects
Tests were carried out to evaluate the toxicity of EN 1-126, EN 1-213, EN 1-215, EN 1-216 and EN 1-218 on beneficial insects (A. mellifera and B. terrestris) by themselves and in combination with two pyrethroids (alpha-cypermethrin (Fastac) and tau-fluvalinate (Mavrik)) and two neonicotinoids (imidacloprid (Confidor) and thiacloprid (Calypso)).

The aim of WP6 was to evaluate the potential of synergist/insecticide applications to select for resistance compared with application of insecticide alone. Selection of a field population of Bemisia tabaci was carried out using alpha-cypermethrin with and without 100 ppm PBO and with thiacloprid with and without 100 ppm PBO. The selection was carried out until January 2015 corresponding to generation 8 of the whitefly population. The total number of generations under selection pressure was 6 generations. At this point final full dose-response bioassays were carried out to assess the susceptibility of the selected and unselected populations. In the case of M. persicae selections were carried out using the same treatment regimes as reported in PR1, these started on July 7th and selection pressure was applied every 2 weeks. A final bioassay was performed on November 26th, 2014.
In the case of both M. persicae and B. tabaci all four treatment regimes resulted in a significant increase in resistance (see Tables 10-12) with higher selection for resistance observed for thiacloprid than alpha-cypermethrin in both cases of insecticides treatment alone and insecticide treatment in combination with PBO.
Interestingly both thiacloprid+PBO and alpha-cypermethrin+PBO gave lower levels of resistance than when insecticide was used alone. For M. persicae this was statistically significant for both compounds but for B. tabaci this finding was only statistically significant for alpha-cypermethrin.
Taken together this finding suggests that when PBO is used in combination with insecticide it may slow the development of resistance, possibly by inhibiting the evolution of P450-mediated and/or esterase-mediated resistance.
Although this is a very interesting finding this experiment was conducted under controlled environment conditions (with a limited number of insects compared to a field scale scenario) and the study needs to be replicated in the field to confirm this.
We used RNAseq to compare global gene expression levels in selected/unselected B. tabaci and M. persicae populations with the RNAseq data received was of excellent quality.
As expected selection of both B. tabaci and M. persicae with thiacloprid and alpha-cypermethrin (both with and without PBO) resulted in measurable changes in gene expression. This included in genes with known roles in insecticide detoxification, such as cytochrome P450s, GSTs, CEs and ABC transporters.
Interestingly in the case of both insect species a much greater number of detoxification genes were upregulated in the strains selected with both insecticide and PBO. This is consistent with previous research which has shown that PBO induces the expression of both P450s and GSTs in Drosophila melanogaster.
In terms of candidate genes that have previously been shown to metabolise the insecticides used in our selection experiments, it was noteworthy that for B. tabaci, selection with thiacloprid (both with and without PBO) resulted in a substantial increase in the expression of the P450 CYP6CM1. This finding is significant as this P450 has previously been shown to detoxify thiacloprid and therefore likely explains, at least in part, the resistance seen in the B. tabaci selected strains. In the B. tabaci cypermethrin selection experiment a strong candidate gene(s) that explained the resistance of the selected strains was not so apparent, particularly in the case of selection with cypermethrin alone. However, CYP6CM1 was again upregulated after selection with cypermethrin+PBO although not at the same levels as selection with thiacloprid. At least three other P450s were overexpressed in this selected strain.
To our surprise very few candidate genes were identified that might explain the difference in the levels of resistance of the B. tabaci strains selected with insecticide alone and those selected with insecticide+PBO with a cuticular protein the only candidate gene overexpressed in the cypermethrin alone selected strain and no strong candidates overexpressed in the thiacloprid alone selected strain.
For M. persicae a much greater number of genes were differentially expressed and a large number of ‘core’ genes were consistently differentially expressed in all selected strains (5199 genes) compared to the non-selected strains. In terms of candidate genes that may explain the resistance phenotype of the selected strains a number of strong candidate genes were highly overexpressed in these strains. These included esterases matching FE4, which is known to have detoxification activity against pyrethroids, an ABC transporter which may play a role in phase III metabolism (excretion of phase I and II metabolites) and several P450s. Interestingly in the thiacloprid selected strains this included the P450 CYP6CY3 which has been shown previously to detoxify neonicotinoid insecticides.
Analysis of M. persicae populations for target-site resistance mechanisms revealed that the frequency of all mutations actually decreased over the course of the experiment in all cases. This was a surprising finding and included the unselected population which also lost most of the target-site mutations (apart from R81T) over the course of the experiment, possible due to fitness penalties associated with these mutations which provided a selective advantage to a clone that did not carry these mutations. Analysis of target-site resistance in B. tabaci showed that the frequency of the L925I kdr mutation increased in frequency but only after selection with cypermethrin. This finding is logical and suggests, as expected, that selection with PBO does not avoid the selection of target-site resistance.

The aim of WP7 was to validate in the field or glasshouse the most effective novel synergist(s) and to check and estimate the scale up of the selected synergists. Based on the results obtained in WP5 and on the chemical feasibility and cost, EN 1-216 and 1-218 were chosen as selected synergists. In particular, tests carried out with thiamethoxam and cypermethrin showed good results on adult B. tabaci (whitefly) at 2/3 and ½ of product field rate.
EN1-218 was the most effective synergist in combination with the actives at 2/3 and 1/2 of the product label rate. The same synergistic efficacy was observed with A. gossypii on lettuce where EN 1-218 had the highest synergistic effect.
The mortality produced by EN1-216 and EN1-218 on A. mellifera was checked in the presence of a neonicotinoid (thiacloprid-Calypso) and a pyrethroid (tau fluvalinate-Mavrik). No significant differences were observed between the tested synergists whilst a difference was observed between Calypso and Mavrik. In particular, no differences were observed between the active ingredient when applied alone and in combination with the novel synergists.
An economic evaluation regarding the possible industrial cost of EN1-216 and EN1-218 was carried out and the industrial cost of the selected synergist is similar to, or slightly less, than the cost of PBO.

Potential Impact:
The main benefits gained from the EcoSyn cooperation, in particular by the involved SMEs, consisted in increased knowledge on optimising the use of novel synergists for insecticide formulations in agriculture and public health ambits. The research activities performed together with the RTD performers and Research SMEs (Other) enabled to meet the know-how needs of all the interested parties and to maximize the project’s results exploitation opportunities. In fact, the direct economic impact of the EcoSyn is represented by the commercial potential of the two novel synergists, developed and tested within the project, and by the patent applications filed by interested SMEs last September 2015. The main economic benefits are due to the share of revenues generated from the future commercial exploitation of the patents and submission to the relevant Regulatory Authorities upon their approval ,according to the agreement set by all parties, and considering the positive global market scenarios for insecticide in agriculture The socio-economic and environmental impacts connected with the application of the new synergists in agriculture and in Public Health, from reduced dosage of active ingredients and enhanced control of resistant pests without adverse effects on beneficial species, might result mainly in increased agricultural production characterised with a low impact on ecosystems.
The EcoSyn SMEs are fully aware of the opportunities that the successful introduction of novel eco-compatible synergists – capable of reducing insecticide resistance on globally important agro pests (as Bemisia tabaci, commonly known as whitefly and Meligethes aeneus, known as pollen beetle), without adverse effects on beneficial species (as bees and bumblebees) - will provide to European farmers, Farmers Associations and other agro-companies in terms of increase of agricultural production with use of less pesticide, and consequently improved production of foods, safer working conditions, whilst protecting the environment.
As for the technological impact, the EcoSyn consortium demonstrated that the structure of the novel synergists could be predicted from structure activity relationship and modelling studies to give higher inhibition of the metabolic enzyme(s). Thus, the insect’s defence to insecticides could be minimised to greatly increase the effect of insecticide application. Clearly this promotes the possibility of controlling even highly insecticide-resistant insects with a field-rate, or lower, application of insecticide. Such a scenario has the potential to decrease the application rates of insecticides with the concomitant environmental benefits. Moreover, project tests showed that novel structures can positively synergize neonicotinoids and pyrethroids against economically important agricultural and household pests. EcoSyn focused also public health aspects and pests (as Musca domestica and Blattella germanica). It is well known that due to the EU Biocidal Product Regulation a major review of the active substances has been undergoing since 1988. While at the beginning more than 1700 actives were notified, by today less than 700 actives are still in the system, all the others were banned from use. This fact radically cuts not only the availability of insecticides, but their efficacy, and furthermore contributes in developing resistance more rapidly. Considering these challenges, EcoSyn project aimed to develop eco-friendly synergists with the purpose of increasing the efficacy of traditional insecticides and possibly decrease their doses. In a great number of public health pests, resistance is being observed. Fleas, bed bugs, cockroaches, fly etc. – which pose serious health threats - all have their insecticide defence mechanisms. Under such circumstances novel synergists may contribute by making the available insecticides more capable and efficacious, or by slowing the development of resistance. The test performed in EcoSyn demonstrated that a few of the chosen novel synergists might possess these capabilities. Therefore, whether the use of the novel synergists, in combination with insecticides, slows the development of resistance will be assessed by further studies (performed outside the project).
Moreover, EcoSyn demonstrated that in the case of two economically important insect pests (M. persicae and B. tabaci) selection with two insecticides (thiacloprid and alpha-cypermethrin) in combination with the synergist piperonyl butoxide (PBO) gave lower levels of resistance than when insecticide was used alone. This finding suggests that when PBO is used in combination with insecticide it may slow the development of resistance, possibly by inhibiting the evolution of P450-mediated and/or esterase-mediated resistance. This is a very important finding with a significant potential impact. If this can be replicated at a field scale it could form the basis of a resistance management strategy to slow the development of resistance. Because there are limited novel insecticides/modes-of action, strategies and tools to prevent or slow the development of resistance are urgently required. Therefore, additional funding to perform the demonstration activities at field scale will be sought by the consortium to confirm this finding as a matter of some urgency.
Following the in-depth lab tests, glasshouse and semi-field trials of identified novel synergists, the EcoSyn consortium decided to proceed with further evaluation of two synergists with the highest industrial potential. The performed analysis confirmed the patentability of the novel structures. IP considerations resulted in three patent applications being filed, enabling the possibility of commercial exploitation. The socio-economic impact, based on a reduction of environment pollution as well as the associate costs would seem positive by allowing a decrease in insecticide dosage but maintaining efficacy with an associated low cost.
The potential commercial exploitation of the EcoSyn results will contribute to the expansion of European agriculture and increase of competitiveness of the EU companies, and export opportunities, against foreign competitors.
The EcoSyn results and finding were duly disseminated to the identified target stakeholders through multiple communications tools and channels. In particular several press releases and thematic articles in scientific and professional magazines have been issued to disseminate project’s work and research results (among all in Pest Management Science, Rostlinolekarm , Agrinotizie, Pest Magazine, AGROW, Chemistry Today, C&I Magazine).
Moreover specific workshops and site visits addressed to farmers, beekeepers and farmers associations were organised by partners to show and explain field experimental conditions and present bioassays results.
The scientific community were informed on the project activities and findings during several international (EU and extra-EU) and national conferences where the EcoSyn was presented through oral speeches, workshops and poster sessions (i.e. Formulation Resistance Challenges in Berlin, Agri – innovation in London, European Congress of Entomology in York, IUPAC International Congress of Pesticide Chemistry in San Francisco, Crop Protection in Southern Britain, SCI'Agriscience Young Researchers Event in London, Resistance 2015 Rothamsted, 6th annual meeting of the European PhD Network in “Insect Science”, Florence and several more).
EcoSyn partners held numerous meetings with the Regulatory Authorities and professional organisations to present results based on laboratory and field trials bioassay and for the evaluation of impact on beneficial insects and environment.
All dissemination outputs are available on the project website:

List of Websites:
EcoSyn project website -
All the project’s open access publications and dissemination materials are available on the following link:

Contact details:
Endura S.p.a.
Dr Valerio Borzatta
+39 051 5281709