Different Population Phenologies of Grapholita molesta (Busck) in Two Hosts and Two Nearby Regions in the NE of Spain.

Grapholita molesta is an important pest of stone and pome fruits. In commercial orchards, integrated pest management programs use pheromone traps to monitor the population dynamics of G. molesta and adjust treatments. Phenology models can be used to forecast the population phenology of pests and to help optimise the time point at which to spray the orchards with insecticides. In the present study, the adult population phenologies of G. molesta in two provinces of north-east Spain were studied, as well as their fit to the phenology model most used in both provinces. Weekly captures of adults in pheromone traps through the season were recorded over 5 y in a large number of commercial orchards, and these data were used to determine the number of generations of G. molesta in each province. The results show significant differences between provinces in the generation time, being 97 degree days (DD) shorter in the Lleida province than in the Girona province. In Girona province, four generations were registered, while five were detected in Lleida. As a result of the differences found, the phenology model was not able to predict precisely the population dynamics in the Girona province.

Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults.

Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated "pools" of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.

The Contribution of Surrounding Margins in the Promotion of Natural Enemies in Mediterranean Apple Orchards.

(1) Habitat management can enhance beneficial arthropod populations and provide ecosystem services such as biological control. However, the implementation of ecological infrastructures inside orchards has a number of practical limitations. Therefore, planting/growing insectary plants in the margins of orchards should be considered as an alternative approach. (2) Here, we assessed the efficacy of a flower margin composed by four insectary plant species (Achillea millefolium, Lobularia maritima, Moricandia arvensis and Sinapis alba), which was placed on an edge of four Mediterranean apple orchards to attract natural enemies of two apple tree aphids (Dysaphis plantaginea and Eriosoma lanigerum). We also characterized the natural enemies present in the aphid colonies. (3) Our results show that the implementation of a flower margin at the edge of apple orchards attracts predators (Syrphidae, Thysanoptera, Araneae, Heteroptera, Coleoptera) and parasitoids. Parasitoids are the main natural enemies present in aphid colonies in our area. (4) The implementation of the flower margins successfully recruited natural enemy populations, and the presence of parasitoids in the surroundings of the orchards increased the parasitism of D. plantaginea colonies.

Determination of the Baseline Susceptibility of European Populations of Cydia pomonella (Lepidoptera: Tortricidae) to Chlorantraniliprole and the Role of Cytochrome P450 Monooxygenases.

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the key pest on pome fruit and walnut orchards worldwide. Its resistance to available insecticides has been widely reported. Chlorantraniliprole is an anthranilic diamide that was introduced in European countries in 2008-2009 and acts by activating the insect's ryanodine receptors. The aims of this study were to determine the baseline susceptibility of European populations of C. pomonella to chlorantraniliprole, to establish the discriminant concentrations (DC) to check the possible development of resistance, and to know the role of cytochrome P450 monooxygenases (P450) in the possible susceptibility decrease of field populations to the insecticide. Ten field populations from Spain along with others were used to calculate the baseline response of larvae to chlorantraniliprole incorporated into the diet. A pooled probit line was calculated, and three DC were established: 0.3 mg a.i./kg (close to the LC50), 1.0 mg a.i./kg (close to the LC90), and 10 mg a.i./kg diets (threefold the LC99). The DC were used to test the susceptibility of 27 field populations from France, Germany, Hungary, Italy, and Spain. The corrected mortality observed in all cases ranged within the expected interval, even with Spanish populations that showed between 12.1 and 100.0% of individuals with high P450 activity. However, the mortality caused by the DC0.3 decreased as the mean P450 activity increased. Field populations resistant to other insecticides were susceptible to chlorantraniliprole. The determined baseline codling moth susceptibility is a valuable reference for tracking possible future alterations in the efficacy of the insecticide.

Monitoring resistance of Cydia pomonella (L.) Spanish field populations to new chemical insecticides and the mechanisms involved.

BACKGROUND: Widespread resistance of Cydia pomonella to organophosphates was demonstrated in populations from the Spanish Ebro Valley area which showed high levels of enzymatic detoxification. To determine the efficacy of new insecticides, neonate larval bioassays were carried out on 20 field codling moth populations collected from three different Spanish apple production areas. Synergist bioassays were performed to determine the enzymatic mechanisms involved. RESULTS: The least active ingredients were methoxyfenozide, with 100% of the populations showing significantly lower mortality than the susceptible strain, and lambda-cyhalothrin, with very high resistance ratios (872.0 for the most resistant field population). Approximately 50% of the populations were resistant or tolerant to thiacloprid. By contrast, tebufenozide was very effective in all the field populations, as was chlorpyrifos-ethyl despite its widespread use during the last few years. Indoxacarb, spinosad and chlorantraniliprole also provided high efficacy, as did emamectin and spinetoram, which are not yet registered in Spain. CONCLUSION: The resistant Spanish codling moth populations can be controlled using new reduced-risk insecticides. The use of synergists showed the importance of the concentration applied and the difficulty of interpreting results in field populations that show multiple resistance to different active ingredients. © 2017 Society of Chemical Industry.

Comparative Effect of Three Neurotoxic Insecticides With Different Modes of Action on Adult Males and Females of Three Tortricid Moth Pests.

Insecticides are the dominant pest management method in fruit and vegetable crops worldwide owing to their quick effect, low cost, and relatively easy application, but they bear negative effects on human health and the environment. Insecticide mode of action (MoA), target species, and sex are variables that could affect insecticide-induced mortality. We recorded the mortality caused by three neurotoxic insecticides with different modes of action (chlorpyrifos [organophosphate, acetylcholinesterase inhibitor], λ-cyhalothrin [pyrethroid, sodium channel modulator], and thiacloprid [neonicotinoid, nicotinic acetylcholinesterase receptor agonist]) applied topically to adult males and females of three economically important tortricid species [Cydia pomonella (L.), Grapholita molesta (Busck), and Lobesia botrana (Denis & Schiffermüller)] that strongly depend on insecticide use for their control. Concentration and dose-mortality curves were recorded at 24 and 48 h postapplication. Large mortality differences between insecticides (maximum 7,800-fold for LD50) were followed by much lower, yet important, differences between species (maximum 115-fold), and sexes (maximum 41.5-fold). Significant interactions between the three factors indicate that they are not independent from each other. Interestingly, with the organophosphate chlorpyrifos, males of the three species were less susceptible than females, which was unexpected, as females are larger than males. Higher female sensitivity to organophosphates has been reported previously but only in G. molesta, not in other moth species. Our results highlight the importance of taking into account sex in dose-mortality studies with adult moths.

Toxicity of allyl esters in insect cell lines and in Spodoptera littoralis larvae.

We investigated the effects of five allyl esters, two aromatic (allyl cinnamate and allyl 2-furoate) and three aliphatic (allyl hexanoate, allyl heptanoate, and allyl octanoate) in established insect cell lines derived from different species and tissues. We studied embryonic cells of the fruit fly Drosophila melanogaster (S2) (Diptera) and the beet armyworm Spodoptera exigua (Se4) (Lepidoptera), fat body cells of the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Coleoptera), ovarian cells of the silkmoth Bombyx mori (Bm5), and midgut cells of the spruce budworm Choristoneura fumiferana (CF203) (Lepidoptera). Cytotoxicity was determined with use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue. In addition, we tested the entomotoxic action of allyl cinnamate against the cotton leafworm Spodoptera littoralis .The median (50%) cytotoxic concentrations (EC50s) of the five allyl esters in the MTT bioassays ranged between 0.25 and 27 mM with significant differences among allyl esters (P = 0.0012), cell lines (P < 0.0001), and the allyl ester-cell line interaction (P < 0.0001). Allyl cinnamate was the most active product, and CF203 the most sensitive cell line. In the trypan blue bioassays, cytotoxicity was produced rapidly and followed the same trend observed in the MTT bioassay. In first instars of S. littoralis, allyl cinnamate killed all larvae at 0.25% in the diet after 1 day, while this happened in third instars after 5 days. The LC50 in first instars was 0.08%. In addition, larval weight gain was reduced (P < 0.05) after 1 day of feeding on diet with 0.05%. In conclusion, the data provide evidence of the significant but differential cytotoxicity among allyl esters in insect cells of different species and tissues. Midgut cells show high sensitivity, indicating the insect midgut as a primary target tissue. Allyl cinnamate caused rapid toxic effects in S. littoralis larvae at low concentrations, suggesting further potential for use in pest control.

Synthesis of allylic trifluoromethyl ketones and their activity as inhibitors of the sex pheromone of the leopard moth, Zeuzera pyrina L. (Lepidoptera: Cossidae).

BACKGROUND: Trifluoromethyl ketones (TFMKs), structurally related to the pheromones, are good inhibitors of pheromone communication in insects. To determine their activity on Zeuzera pyrina L. (Lepidoptera: Cossidae), a polyphagous pest, the authors have prepared two diunsaturated TFMK analogues of the major (3) and the minor (4) pheromone components, and two monounsaturated ones (5, 6). Their biological activity in electroantennogram (EAG), wind tunnel and field tests is presented. RESULTS: The synthetic strategy to obtain the allylic TFMKs 3 and 5 is based on the reactions of diene 10 and 1-octadecene with trifluoroacetaldehyde ethyl hemiacetal, followed by Dess-Martin oxidation of the resulting homoallylic trifluoromethyl alcohols. In EAG, topical application of analogues 3 and 4 on male antennae significantly reduced the pheromone response. In the wind tunnel, compound 4 reduced the number of contacts with the pheromone source. In the field, traps baited with mixtures of pheromone and inhibitors captured significantly fewer males than the pheromone alone. CONCLUSION: An efficient synthesis of allylic TFMKs is reported, with good overall yield, regiospecificity and diastereoselectivity. These compounds are good inhibitors of the pheromone in electrophysiology, wind tunnel and field tests. The results show the importance of two unsaturations at positions 2 and 13 of the trifluoroacyl group in the structure of the analogues, the latter being critical for inhibitory activity.

Ordinary glomeruli in the antennal lobe of male and female tortricid moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae) process sex pheromone and host-plant volatiles.

Both sexes of Grapholita molesta, a key pest of stone fruits, are able to detect host-plant volatiles and the sex pheromone emitted by females, and to modify their behaviour accordingly. How olfactory information is processed in the central nervous system is unknown. Intracellular recordings and stainings were used to characterize antennal lobe (AL) neuron responses to single pheromone components, a behaviourally active blend of five peach volatiles and a pear-fruit ester. AL neurons with different response patterns responded to pheromone components and plant volatiles. In males more neurons responded specifically to the main pheromone component than in females, whereas neurons responding to all three pheromone components were more abundant in females. Neurons responding to all three pheromone components often responded also to the tested plant volatiles in both sexes. Responses to all pheromone components were dose dependent in males and females, but dose-response relationships differed between neurons and tested pheromone components. Among the five AL projection neurons identified neuroanatomically in males, no arborizations were observed in the enlarged cumulus (Cu), although all of them responded to pheromone compounds. In one of two stained projection neurons in females, however, the glomerulus, which is thought to be homologous to the Cu, was targeted. The processing of pheromone information by ordinary glomeruli rather than by the macroglomerular complex is thus a striking feature of this species, indicating that pheromone and plant volatile processing are not entirely separate in this tortricid moth AL. However, the absence of recorded pheromone responses in the Cu needs to be confirmed.

Susceptibility to organophosphate insecticides and activity of detoxifying enzymes in Spanish populations of Cydia pomonella (Lepidoptera: Tortricidae).

The mechanisms associated with resistance of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), to organophosphate insecticides in pome fruit orchards have been shown to depend on the area. Our objectives were to evaluate the susceptibility of Spanish codling moth populations to chlorpyrifos-ethyl, azinphos-methyl, and phosalone, and the activity of three enzymatic systems reported to be involved in resistance. Eleven field populations and a susceptible strain used as a reference were tested using a bioassay consisting in the topical application of a diagnostic concentration on postdiapausing larvae. The enzymatic activity of mixed function oxidases (MFO), glutathione transferases (GST), and esterases (EST) was measured in postdiapausing larvae and adults. A significant decrease in the efficacy of the organophosphates was observed for all field populations, although the decrease was smaller in the case of chlorpyrifos-ethyl. No differences between sexes were detected. In postdiapausing larvae, the activity of the three enzymatic systems was higher in all the field populations than in the susceptible population. The possible implication of EST in codling moth insecticide resistance is reported for the first time in European field populations. In adults, only MFO and GST were implicated. Codling moth resistance to organophosphates in Spanish populations must be taken into account in the implementation of antiresistance strategies.

Three-dimensional antennal lobe atlas of the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae): comparison of male and female glomerular organization.

The oriental fruit moth Cydia molesta is an important pest and the behavioural role of olfactory signals such as pheromones and plant volatiles have been studied extensively in both sexes. To understand odour processing further, however, detailed knowledge of the anatomy of the olfactory system is crucial. In the present study, an atlas of the antennal lobe (AL) is presented based on the three-dimensional reconstructions of both ALs of three male and three female brains by means of neuroanatomical and computational approaches. We identified 48-49 "ordinary" glomeruli and one large glomerulus situated at the entrance of the antennal nerve in males, and 49-52 "ordinary" glomeruli and one large glomerulus in the ventro-medial part of the AL in females. Anomalous supernumerary, anomalous missing and sexually dimorphic glomeruli were found in the studied individuals in greater numbers than in other lepidopteran species. Male and female maps were compared with respect to glomerular size and position with 45 glomeruli being matched, indicating a conserved glomerular pattern between the sexes. Three additional glomeruli were sexually dimorphic in size and five male-specific and six female-specific glomeruli were also found. Palp backfills resulted in the staining of a unique glomerulus in both sexes identified as the sexually dimorphic glomerulus 45. This glomerulus was never stained from antennal backfills, which stained the other glomeruli of the AL. The three-dimensional atlas can now be used to elucidate the functional role of individual glomeruli in both sexes of C. molesta.

Synthesis of allyl esters of fatty acids and their ovicidal effect on Cydia pomonella (L.).

Eight allyl esters of fatty acids were synthesized in moderate to high yields with a novel two-step procedure using glycerol as a starting material. The two-step methodology avoids the use of allyl alcohol. The first step consisted of heating at 80 degrees C for 48 h a 2:1:5 mmol mixture of glycerol, a fatty acid, and chlorotrimethylsilane in a solvent-free medium. The crude compound was then dissolved in butanone and heated at 115 degrees C in the presence of NaI. A tandem Finkelstein rearrangement-elimination reaction occurs, producing the corresponding allyl ester. The activity of these esters against Cydia pomonella (L.) (Lepidoptera: Tortricidae) eggs was tested in the laboratory by topical application of one 0.1 microL drop. All of the compounds showed a concentration-mortality response and caused 100% mortality at the highest concentration tested (10 mg/mL). There was an inverse relationship between the alkyl chain length and the ovicidal activity of the allyl ester; the LC(50) and the LC(90) of the two compounds that have the longer alkyl chains were significantly higher than those of the rest of the compounds. The ovicidal and IGR activities of this kind of compound appear to be unprecedented.

Diurnal variation of walnut tree volatiles and electrophysiological responses in Cydia pomonella (Lepidoptera: Tortricidae).

BACKGROUND: In recent years, many studies have been carried out on the behavioural and electrophysiological responses of Cydia pomonella (L.) to host volatile emissions, to find alternative attractants to the sex pheromone for pest monitoring. These studies have focused on apple and pear, and very little has been done on walnut. In the present work, the diurnal and seasonal variation in walnut volatile emissions and the electrophysiological response of C. pomonella have been studied. RESULTS: Ninety compounds were detected in walnut emissions, mainly monoterpenes and sesquiterpenes. The most abundant compound was beta-pinene, which, together with (Z)-3-hexenyl acetate, (E)-beta-ocimene, limonene, germacrene D, 1,8-cineole, sabinene, (E)-beta-farnesene, (E)-beta-caryophyllene, beta-myrcene and beta-phellandrene, constituted between 81.9 and 90.5% of the total chromatographic area. Differences between seasonal periods were significant for 39 compounds, and between daytimes for 14 compounds. Discernible and consistent EAD responses were detected to 11 walnut-origin compounds, and confirmed with synthetics to seven of them. Except for alloocimene, pinocarvone and caryophyllene oxide, all these compounds are also emitted by apple. CONCLUSION: Walnut volatile emissions differ widely from apple ones, but both share many compounds that are EAD-active in C. pomonella. However, among EAD-active compounds there are three walnut-specific ones, which should be further tested in behavioural assays.