Sublethal Effects of Neonicotinoid Insecticide on Calling Behavior and Pheromone Production of Tortricid Moths

In moths, sexual behavior combines female sex pheromone production and calling behavior. The normal functioning of these periodic events requires an intact nervous system. Neurotoxic insecticide residues in the agroecosystem could impact the normal functioning of pheromone communication through alteration of the nervous system. In this study we assess whether sublethal concentrations of the neonicotinoid insecticide thiacloprid, that competitively modulates nicotinic acetylcholine receptors at the dendrite, affect pheromone production and calling behavior in adults of three economically important tortricid moth pests; Cydia pomonella (L.), Grapholita molesta (Busck), and Lobesia botrana (Denis & Schiffermüller). Thiacloprid significantly reduced the amount of calling in C. pomonella females at LC0.001 (a lethal concentration that kills only 1 in 105 individuals), and altered its calling period at LC1, and in both cases the effect was dose-dependent. In the other two species the effect was similar but started at higher LCs, and the effect was relatively small in L. botrana. Pheromone production was altered only in C. pomonella, with a reduction of the major compound, codlemone, and one minor component, starting at LC10. Since sex pheromones and neonicotinoids are used together in the management of these three species, our results could have implications regarding the interaction between these two pest control methods.

in the management of these three species, our results could have implications regarding the interaction between these two pest control methods.

INTRODUCTION
In lepidopterans, reproduction shows a periodic pattern related to the duration of the daily light and dark cycles and involves a complex series of behavioral and physiological events including chemical communication mediated by sex pheromones (Groot 2014).Usually females release the sex pheromone and males fly towards females from tens or hundreds of meters (Cardé 2016).
Closely related moth species with common phylogenetic origins are under competition for limited communication channels (Roelofs and Brown 1982).Reproductive isolation is instrumental in speciation (Smadja and Butlin 2009), and in the case of pheromone communication is modulated by species-specific differences in sex-pheromone composition and time of release (Byers 2006;Groot 2014) Several factors influence calling behavior and pheromone production in moths (McNeil 1991;Raina 1993), including age (i.e., Webster and Cardé 1982;Gemeno and Haynes 2000;Kawazu and Tatsuki 2002;Mazor and Dunkelblum 2005;Ming et al. 2007), mating status (i.e., Foster and Roelofs 1994;Delisle et al. 2000;Mazo-Cancino et al. 2004), and pheromone autodetection (Holdcraft et al. 2016).Environmental stressors, such as sublethal doses of insecticides that intoxicate but do not kill the individual, could also affect pheromone production and release (Haynes 1988;Tricoire-Leignel et al. 2012), but this aspect has been tested in relatively few moth species.
Pesticides are often considered a quick, easy, and inexpensive solution to control insect pests.
However, pesticides can cause negative effects on the environment and human health (Aktar et al. 2009).In Integrated Pest Management (IPM) the use of insecticides is often combined with environmentally safer methods (Damos et al., 2015), such as the use of sex pheromones for mating disruption (emitting large amounts of synthetic sex pheromone and so reducing the probability of mate finding), mass trapping (removing from the population individuals attracted to traps baited with pheromone lures), and monitoring the population for precise timing of control procedures (Witzgall et al. 2010).Because semiochemicals exploit insect chemical communication, and neurotoxic insecticides affect the normal functioning of the nervous system, it is plausible that the simultaneous use of semiochemicals and insecticides could affect each other in IPM strategies (Suckling et al. 2016).Indeed, several studies report alterations of the normal perception of and response to chemical signals in insects treated with sublethal doses of insecticides (Haynes 1988;Tricoire-Leignel et al. 2012).
In this context of potential semiochemical and toxicological interactions in the agroecosystem, we explored the effect of sublethal doses of a neonicotinoid insecticide on pheromone production and release (i.e., calling behavior) in three tortricid moths.Our test species, Cydia pomonella (L.), Grapholita molesta (Busck) and Lobesia botrana (Denis & Schiffermüller), are main pests of apple, peach and grapevines, respectively, have a relatively worldwide distribution and are controlled with both semiochemicals and insecticides (Ioriatti et al. 2011;Kirk et al. 2013;Damos et al. 2015).Indeed these three species represent several of the most successful examples of pest control by means of mating disruption (Witzgall et al. 2010).For a toxicant, we used the neuroactive insecticide thiacloprid, a neonicotinoid that competitively modulates nicotinic acetylcholine receptors at the dendrite (Casida 2009).Thiacloprid is recommended for the control of C. pomonella and G. molesta in stone and pome fruits in Spain (Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente [MAPAMA], 2017).Although aimed at eggs and larvae, thiacloprid residues from air blast spraying in fruit orchards could potentially intoxicate adults with residual sublethal doses, and therefore affect semiochemical control (Wise et al. 2006).Thiacloprid could potentially affect L. botrana in vineyards adjacent to fruit orchards treated with this insecticide (Harari et al. 2011).Baseline mortality with thiacloprid has been determined for the three species under laboratory conditions (Navarro-Roldán et al. 2017).
Cydia pomonella and G. molesta belong to the tribe Grapholitinii and L. botrana to the tribe Olethreutini, both in the subfamily Oletheutrinae (Regier et al. 2012).By comparing the effect of thiacloprid across phylogenetically related species of diverse ecology we hoped to gain basic background information about sublethal effects of neurotoxic insecticides on sex pheromone signalers.

METHODS AND MATERIALS
Insects.Susceptible laboratory strains of C. pomonella, G. molesta and L. botrana established from individuals collected in Lleida (Spain), Piacenza (Italy), and La Rioja (Spain), respectively, have been maintained under laboratory conditions for more than 5 years without introduction of wild individuals.Larvae were reared in artificial diet (Ivaldi-Sender 1974) at 25 ± 1 ºC under a 16:8 h light:dark photoregime.Females of G. molesta and L. botrana were separated at the pupal stage and adult emergence was checked daily and always at the same hour.C. pomonella was sexed at the adult stage, also in a daily basis.Because adults were collected only once per day, they were 0-24 h old when separated from the pupae, 24-48 h old one day later, and so on.
Treatments were applied during the first half of the photophase at 0-24 h post-emergence (calling behavior test), or at 16-40 h post-emergence (pheromone gland test).One to three adults were placed in 10 ml clear polystyrene test tubes and received a brief (10 sec) flow of industrial grade CO2 which quickly anesthetized them.Immediately after being anesthetized they were placed upside down under the field of view of a stereo microscope and a 1 µl test solution was applied to the ventral thoracic region of each individual using a high-precision, positive displacement, repeatable-dispensing micropipette (Multipette ® -M4, Eppendorf, Germany).Treated females were transferred immediately to a 150 ml polypropylene non-sterile clinical sample bottle (57 mm diameter x 73 mm-high).Individuals receiving the same treatment were placed in groups of 3 to 10 in the same bottle.The lid of the bottle was punctured to make 10 1-mm-diameter holes to allow gas exchange, and a 1.5 ml Eppendorf tube ® containing 10% sugar solution and cotton lid was placed on the bottom to supply nutrients.Bottles with treated insects were placed in the rearing room until test time.
Mortality was determined 24 h post-treatment.Adults were observed with the naked eye and scored as: 1) alive if they flew or walked apparently unaffected; 2) as potentially moribund if they could barely walk or were laying on the floor of the bottle but still moved; 3) or as potentially dead if they laid immobile on the floor of the bottle.Mortality was estimated using the sum of the potentially moribund and dead individuals.The other individuals scored as alive were used in the calling and pheromone tests.No further anesthesia was needed.
Calling Behavior.Females were placed individually in 9 mm-long x 1.5 mm-diameter, 10 mL clear polystyrene test tubes that had both ends covered with 1.5 mm-diameter-mesh galvanized wire screen (Figure S1).Tubes were placed on a 42 cm-tall platform that could hold up to 13 tubes from bottom to top, leaving 2.5 mm between them (Figures S1 and S2).The platforms were painted white to facilitate observation of calling postures inside of the plastic tubes.Four platforms with test tubes were placed in a chamber with a continuous 0.4 ± 0.1 m s -1 air flow.
The tubes were aligned with the air flow (flow through the tubes was not measured) to minimize ambient pheromone levels, which could affect moth calling behavior (Holdcraft et al. 2016).
Four 18-watt domestic fluorescent lamps (Standard daylight F18W/154-T8, Sylvania) that were placed between 20 and 52 cm above the highest and lowest female positions in the rack provided between 4700 and 1700 lux during photophase, respectively (TES-1330, Tes Electrical Electronic Corp.).During scothophase there was complete obscurity and calling was observed using a 660-nm LED (2.5V, 1.3 candles, 5 mm diameter, 30° view angle, LedTech, part number LURR5000H2D1) which was held manually near each female for observations.G. molesta females call mainly before the beginning of the scotophase, C. pomonella females call mainly during the scotophase (Groot 2014), and L. botrana females call during the first hours of scotophase (Harari et al. 2011(Harari et al. , 2015)).However several factors (i.e., illumination, temperature etc.) could affect the calling period (McNeil 1991), so in order to determine the exact calling period of our laboratory colonies under our experimental conditions we performed preliminary observations on 69-75 untreated (i.e., no acetone or insecticide) individuals of each species over a 12-h period bracketing the expected calling times.The 12-h observation periods of C. pomonella and L. botrana started 2 h before the onset of the scotophase, covered all the scotophase and continued during the first 2 h of the photophase.The observation period of G. molesta started 8 h before the onset of the scotophase and extended 4 h into the scotophase.In order to observe the three species during the same 12 h time period, the photoregimes of C. pomonella and L. botrana were synchronized with each other and both were observed on the same day, the photoregime of G. molesta was delayed with respect to that of the other species.
Observations of the two groups were made on alternate days, and were performed at 30 min intervals, except during the last 30 min of the photophase and the first 2 h of the scotophase when they were observed every 15 min to increase sample resolution for the relatively short (about 2 h) calling period of L. botrana.Females were placed in the observation setup at least 30 min before the first observation.The first observations during scotophase occurred between 5 to 10 minutes after lights off.
Once the calling period of our laboratory insects was determined (Figure S3), between 61 and 70 females treated with sublethal insecticide doses or acetone were observed during the same period as in the preliminary test to determine the effect of the insecticide dose on calling behavior.
Calling behavior was categorized as either "weak calling" (the female walks or is slightly agitated, with an intention to adopt, or beginning to adopt, a calling posture consisting in rising its wings and extruding the abdomen tip), "medium calling" (incomplete calling stance: more or less stationary female with partially raised wings and abdomen tip partially extruded), or "strong calling" (full calling stance: mostly stationary female with fully raised wings, and protruded abdomen tip readily visible).The specifics of the calling posture were slightly different and characteristic across species.
Pheromone Gland Content.Pheromone was extracted from females that were 40-to 64-hour-old and had been treated with sublethal insecticide doses, or acetone as control, 24 h earlier.
Extractions were restricted to a 1 h period coinciding with peak calling time of each species: 30 to 90 min after the onset of scotophase in C. pomonella, 120 to 60 min before the scotophase in G. molesta and 0 to 60 min after the onset of scotophase in L. botrana.The tip of the abdomen containing the sex pheromone gland tissue was excised carefully by pulling it from the abdomen with fine forceps.Abdominal tips were deposited individually in solvent-rinsed and oven-dried conical-bottom glass vials (Total recovery vial, part number 186002805, Waters, USA) with Teflon-lined lids (part number 186000274, Waters, USA) containing 7 μl of a 1ng/µl octadecane internal standard solution (> 99% pure, Sigma-Aldrich, Spain) in n-hexane (> 97% pure, VWR Chemicals, BDH-Prolabo, Spain).After 30 min at room temperature the glands were removed from the vial and the extracts were stored at -20°C until analysis (for a maximum of 10 days).
The remaining extract (approx.0.5-3 μl) was injected in a Hewlett Packard 6890 gas chromatograph equipped with a flame ionization detector and a 30 m-long, 0.25-mm I.D., 0.25μm film-thickness DB-Wax column (Agilent Technologies, Madrid, Spain).The constant helium flow through the column was 1 ml min -1 , and the injector and detector temperatures were 250 and 270°C, respectively.The oven temperature program stayed at 70ºC during 1 min and then increased to 170°C at 30°C min -1 , and from 170°C to 230°C at 10°C min -1 , and remained at this molesta and E,Z-7,9-12:Ac in L. botrana) were calculated.
Statistical Analyses.All the statistical analyses were run in R software (R Core Team 2016).
Mortality was analyzed with Fisher's exact tests and Bonferroni correction.To determine the effect of thiacloprid on the calling period, we calculated the first, mid and final times of calling for calling females.To determine the effect of thiacloprid on the amount of calling behavior we calculated the proportion of observations in which females called out of the total number of observations of the calling period estimated previously.For example, for an 8-h calling period and observations every 30 min there would be 960 observations for 60 insects.If calling appeared in 480 of these observations, then the amount of calling would be 50%.Analyses were performed with generalized linear models (GLM), using Gaussian family functions for continuous variables (calling period and pheromone composition) and binomial family functions for binomial variables (amount of calling).The predictmeans() function performed Tukey´s multiple pairwise comparisons and provided parameter estimates and their standard errors and confidence intervals which are shown in tables and figures.Raw data and R scripts are available online (http://hdl.handle.net/10459.1/59531).Whenever the term "significant" is used in the text regarding differences between treatments it indicates a p-value < 0.05.S1) was comparable to the dose-mortality curves used to determine the test concentrations (Navarro-Roldán et al. 2017).Acetone and LC0.001 did not induce any mortality, and the maximum mortality with LC1 was below 2.5 %.LC20 mortality ranged between 7% and 21%, and with LC10 it was between LC1 and LC20 in all but one case (Table S1).

Mortality in our tests (Table
Calling Behavior.Under our test conditions, C. pomonella, G. molesta and L. botrana had distinct calling periods.C. pomonella called throughout the scotophase, G. molesta called from 4 h before the start of the scotophase to 0.5 h into the scotophase, and L. botrana called for 2.5 h starting at the beginning of the scotophase (Figure S3).Acetone did not appear to affect the amount or periodicity of calling with respect to untreated females (compare Figure 1 and Figure S3).At least 80% of the acetone-treated females called during peak calling time (all species), but there was a significant dose-dependent reduction of calling in treated females (Figure 1).In C. pomonella there was a strong reduction on the amount of calling which was already significant at the lowest concentration (LC0.001), in the other two species the reduction started with LC1 (Table 1), and although significant, the effect was very mild in L. botrana.Peak calling reductions with LC20 were 70.19 and 75.09 % for C. pomonella and G. molesta, respectively.In L. botrana calling was not reduced beyond LC1, and reduction with respect to the control treatment was only 10%.A small percentage (< 8 %) of the control females did not call a single time during the entire observation period, but this number increased with thiacloprid doses and peaked at LC20 with 53% (C.pomonella), 61% (G.molesta) and 20% (L.botrana) non-calling females, respectively (Table S2).Individual differences in the number of calling observations per female and intensity of calling (weak, medium and strong) were observed (Figures S4, S5 and S6, data not analyzed).In general, "strong" calling coincided with peak calling time, whereas "weak" calling appeared to increase with insecticide dose.
Sublethal doses of thiacloprid modified calling periods (Table 2).LC1, LC10 and LC20 advanced the end and midpoint calling times of C. pomonela's (150 min, approx.),and delayed the start and midpoint calling times of G. molesta (74 min, approx.).No significant effect in calling period was observed in L. botrana.Pheromone Gland Content.The two highest sublethal doses of thiacloprid, LC10 and LC20, reduced significantly the quantity of the major pheromone component of C. pomonella (codlemone, E,E-8,10-12:OH) from about 5 ng to about 2 ng, and the minor component 12:OH from about 2 ng to about 1 ng, whereas the other two minor components of C. pomonella and the pheromone components of the other two species were unaffected (Figure 2).Reduction in the quantity of the major pheromone component of C. pomonella resulted in an increase in the relative proportion of two minor compounds, E9-12:OH and 14:OH, relative to codlemone (Table 3).This effect was significant only at the highest pheromone dose, LC20.E9-12:Ac and 14:OH were 14 and 16% relative to codlemone in acetone control females, and 56 and 40% relative to codlemone in LC20 thiacloprid treated females.No further changes in the proportion of pheromone components were observed in C. pomonella or the other two species.

DISCUSSION
Thiacloprid persists as surface residue on fruit and leaves (Wise et al. 2006), and has a half-life in the soil of 10 to16 days (Krohn 2001).Therefore, adult moths could be exposed to sublethal doses of thiacloprid even though the application is not aimed at them but to other life stages, or even at other pest species, or from drift by blast sprayers in neighbor fields.In the present study, sublethal doses of thiacloprid producing as low as 0.001 mortality significantly modified female pheromone signaling, but the effect was not the same on the three tortricid species.
In our study C. pomonella called throughout the scotophase as previously reported (Castrovillo and Cardé 1979;Weissling and Knight 1996).Reports on the calling period of G. molesta are only slightly different from ours (Baker and Cardé 1979;Stelinski et al. 2006Stelinski et al. , 2014)), which could be explained by the effect that variations in light and temperature have on the calling period of moths (Baker and Cardé 1979;Castrovillo and Cardé 1979;Groot 2014).To our knowledge, our study provides the first complete observation on the calling period of L. botrana.
Its onset of calling coincides with a previous report (Harari et al. 2015).Regarding pheromone gland composition, our estimations are generally similar to what has been previously reported (summarized in Table S3).Minor differences across studies could be attributed to population differences or to methodological aspects related to the extraction and analysis of compounds that are present in very low quantities in the pheromone glands.In general, the mortality caused by thiacloprid was similar to the expected levels of mortality estimated in a previous study (Navarro-Roldán et al. 2017).
The most dramatic phenotypic effect of sublethal thiacloprid doses in our test species was the significant reduction in the amount of calling in C. pomonella females treated with LC0.001, a remarkably low concentration that kills only one in 10 5 females.The other two species were less sensitive, and the effect on L. botrana, although statistically significant was so mild that probably would not have a real effect in the field.The calling curves of the insecticide treatments for the most part fell within the boundaries of the acetone control curves, so the shift in calling period with thiacloprid was not as remarkable as the effect on the amount of calling.A detrimental effect of sublethal insecticide on calling behavior has been observed in other moth species with pyrethroid (Haynes and Baker 1985;Clark and Haynes 1992a;Yang and Du 2003;Shen et al. 2013;Quan et al. 2016) and organophosphate insecticides (Trimble et al. 2004).
Insecticides do not always decrease calling behavior, as in the case of Ostrinia furnacalis (Güenee) and Spodoptera litura (Fabricius) treated with pyrethroids as larvae (Wei and Du 2004;Wei et al. 2004).Yet, sublethal insecticide could increase the percentage of calling females, as in Trichoplusia ni (Hübner) treated with chlordimeform (Clark and Haynes 1992b).Regarding the timing of calling behavior, Haynes and Baker (1985) observed that for their highest permethrin dose (15 ng/moth, approx.an LC10) the end of the calling period of Pectinophora gossypiella (Saunders) was reduced by 1 h.Surviving adults of O. furnacalis (Wei and Du 2004) and Choristoneura fumiferana (Clemens) (Dallaire et al. 2004) larvae treated with deltamethrin and tebufenocide, respectively, started to call 1 h later than control females.
The calling periods that we have observed in tortricids under laboratory conditions may be different under natural light conditions because our laboratory photoregime did not provide the smooth light:dark transition that occurs at dawn and sunrise in the field, and this factor alone is known to affect the periodicity of locomotor activity of other insects (Vanin et al. 2012).
Captures of male C. pomonella in pheromone traps show a 4-h activity peak around dusk time under natural conditions (Knight et al. 1994), which suggests that the relatively long calling period of C. pomonella observed under artificial conditions could be narrower under more natural light conditions.Unlike calling behavior, thiacloprid only affected pheromone production in one of the three species, C. pomonella, and it required higher doses than what was needed to affect calling behavior.The quantity of the major compound, codlemone, and one of the three minor compounds, 12:OH, were approximately halved compared to the acetone control at LC10 or LC20, and the ratio with respect to codlemone of two minor compounds, E9-12:OH and 14:OH, increased 4 and 2.5-fold, respectively, at LC20.Detrimental effects on pheromone production have been observed with deltamethrin in O. furnacalis (Yang and Du 2003) and with azinphosmethyl in Choristoneura rosaceana (Harris) (Delisle and Vincent 2002;Trimble et al. 2004).
Changes in component ratios with sublethal doses of deltamethrin have been described in S. litura (Wei et al. 2004), and with a biopesticide mixture containing Bacillus thuringiensis (Berliner) and abamectin in H. armigera (Shen et al. 2013).Lack of effect of sublethal doses on pheromone production, as in G. molesta and L. botrana, has been described also in T. ni treated with cypermethrin and chlordimeform (Clark and Haynes 1992a,b).
It is interesting that thiacloprid affected calling behavior and pheromone production in C. pomonella but only calling behavior in G. molesta and L. botrana.In other species there is also a differential effect of insecticide on calling behavior and pheromone production (Clark and Haynes 1992a,b;Yang and Du 2003;Trimble et al. 2004;Wei and Du 2004;Shen et al. 2013).
Pheromone biosynthesis in moths is mediated by a brain-released neurohormone (PBAN) that reaches the pheromone gland through the haemolymph and binds to specific receptors on the membrane of pheromone secretion cells (Jurenka and Rafaeli 2011;Groot 2014).A likely mechanism by which the neurotoxic insecticide thiacloprid could alter pheromone production is by reducing PBAN secretion.In O. furnacalis an homogenate of the PBAN-producing tissues from females treated with the pyrethroid deltamethrin, which produced less pheromone than controls, resulted in a reduction of pheromone titer in the glands of the decapitated females in which it was injected, which suggests that deltamethrin reduced PBAN secretion in this species (Yang and Du, 2003).It appears that juvenile hormone (JH) is involved in the regulation of calling behavior (Rafaeli 2009), and therefore insecticides may affect calling behavior and pheromone production differently.Since PBAN, JH and pheromone biosynthesis mechanisms are probably very similar in the three tortricid species (Roelofs and Rooney 2003;Jurenka and Rafaeli 2011), it remains to be determined why similar sublethal doses of thiacloprid resulted in differential effects in pheromone production and calling behavior among the three moth species.
Several questions need to be solved in order to determine the impact of our findings in IPM control.Males respond not to the pheromone in the gland but to the volatiles released by calling females, so we need to know if thiacloprid alters the composition of the pheromone blend emitted by females, as has been reported in T. ni with chlordimeform (Clark and Haynes, 1992b).
Obviously the effect of thiacloprid on male response needs to be determined too, as insecticides are known to affect moth pheromone responses (Linn and Roelofs 1984;Wei and Du 2004;Wei et al. 2004;Zhou et al. 2005;Knight and Flexner 2007;Rahbi et al. 2016).Additionally, it needs to be determined if thiacloprid-treated females are as attractive to males as untreated ones, or less active at mating than untreated ones, as has been shown in other moth species (Delpuech et al. 1998;Wei et al. 2004;Knight and Flexner 2007;Reinke and Barrett 2007;Barrett et al. 2013;Quan et al. 2016).Mating in our test species is preceded by a courtship that may include contact chemical cues and short-range pheromones associated with male hair pencil displays (Jurenka and Rafaeli 2011), and these elements of mating behavior could also be affected by thiacloprid.
If thiacloprid is detrimental to these elements of mating behavior, its effect on reproduction may be even larger than what our results suggest, with a possible enhancement of semiochemical IPM control.For this reason, basic knowledge of insecticide effects on insect behavior, physiology, and reproductive success could be a critical issue if we want to optimize IPM strategies.

Figure 1
Figure 1 Effect of thiacloprid on the percentage of females calling (N=61-70).The grey area

Figure 2
Figure 2 Effect of thiacloprid on the quantity of individual pheromone components in the

Table 1
Effect of thiacloprid on the percentage of calling observations during the calling period.Different letters within a species indicate 534 significant differences among insecticide treatments (P<0.05,Tukey after GLM).
a N ♀ = number of females; N Obs.= number of observations into the calling period of each species; N Tot.= total N consider in GLM analysis, which is the product between 536 N♀ and N Obs.537 538

Table 2
Effect of thiacloprid on the start, mid and end calling times relative to the onset of the scotophase (in minutes).Different letters within a 539 column and species indicate significant differences among treatments (P < 0.05, Tukey after GLM).N = number of females.