Chemical and Electrophysiological Characterisation of Headspace Volatiles from Yeasts Attractive to Drosophila suzukii.

Chemical control of Drosophila suzukii (Diptera: Drosophilidae) based on the use of insecticides is particularly challenging as the insect attacks ripening fruits shortly before harvest. An alternative strategy may rely on the use of yeasts as phagostimulants and baits, applied on canopy as attract-and-kill formulations. The aim of this research was to identify the most attractive among six yeast species for D. suzukii: Saccharomyces cerevisiae, Hanseniaspora uvarum, Clavispora santaluciae, Saccharomycopsis vini, Issatchenkia terricola, and Metschnikowia pulcherrima. The volatile profile of C. santaluciae was described for the first time. Behavioural experiments identified H. uvarum and S. vini as the most attractive yeasts. The characterization of yeast headspace volatiles using direct headspace (DHS) and solid-phase microextraction (SPME) revealed several strain-specific compounds. With DHS injection, 19 volatiles were characterised, while SPME revealed 71 compounds constituting the yeast headspace. Both analyses revealed terpenoids including ß-ocimene, citronellol, (Z)-geraniol (nerol), and geranial as distinct constituents of S. vini. H. uvarum and S. vini were further investigated using closed-loop stripping analysis (CSLA) and electroantennography. Out of 14 compounds quantified by CSLA, ethyl acetate, isoamyl acetate, ß-myrcene, benzaldehyde and linalool were detected by D. suzukii antennae and might generate the strong attractiveness of S. vini and H. uvarum. Our results highlight a strong attraction of D. suzukii to various yeasts associated with both the flies and their habitat and demonstrate how different sampling methods can impact the results of volatile compound characterization. It remains to be demonstrated whether the distinct attraction is based on special adaptations to certain yeasts and to what extent the metabolites causing attraction are interchangeable.

Diel rhythm of volatile emissions from males and females of the olive fruit fly Bactrocera oleae using PTR-ToF and GC-MS.

The olive fruit fly Bactrocera oleae, is the major key pest of olive groves worldwide. As an odor-driven species, its intraspecific communication has been thoroughly investigated, yielding a combination of spiroacetals, esters and hydrocarbons. However, its management with pheromone is still restricted to olean, the major pheromone component. Given the crucial role of circadian rhythm and pheromone blends in mediating flies reproductive behavior compared to single compounds, B. oleae headspace chemical profile was carefully examined, through the combination of Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToF) and Gas Chromatography coupled with Mass Spectrometry (GC-MS). This novel approach aimed at continuously investigating the temporal scale of volatilome profile of B. oleae individuals, as well as the determination of new candidate sex-borne compounds (particularly those emitted in traces or having low molecular weight), that may be relevant to the fly's chemical communication and were unreported due to limitations of frequently used analytical techniques. Our results describe the dynamics and diversity of B. oleae chemical profile, highlighting the emission of 90 compounds, with clear diel rhythm of release, of known pheromone components of B. oleae (e.g., olean, alpha-pinene and muscalure) and new candidates. In contrast to ammonia, acetaldehyde and muscalure, which were highly emitted during the afternoon by males and mixed groups, olean was mostly released by mature females and mixed groups, with a peak of emission during early-morning and afternoon. This emission of olean around dawn is reported for the first time, suggesting early-morning mating activity in B. oleae. Furthermore, esters, such as methyl tetradecanoate, which had been earlier identified as a pheromone for B. oleae, did not exhibit any discernible release patterns. These findings are the first to demonstrate the emission of chemicals, which are only produced when males and females are close to one another, with an emission peak during the afternoon (mating period), and that may have aphrodisiac properties for B. oleae males. These results emphasize the relevance of compounds with distinct diel rhythm and address their potential function as intraspecific messengers, according to their source and timing of release.

Combined effect of a neonicotinoid insecticide and a fungicide on honeybee gut epithelium and microbiota, adult survival, colony strength and foraging preferences.

Fungicides, insecticides and herbicides are widely used in agriculture to counteract pathogens and pests. Several of these molecules are toxic to non-target organisms such as pollinators and their lethal dose can be lowered if applied as a mixture. They can cause large and unpredictable problems, spanning from behavioural changes to alterations in the gut. The present work aimed at understanding the synergistic effects on honeybees of a combined in-hive exposure to sub-lethal doses of the insecticide thiacloprid and the fungicide penconazole. A multidisciplinary approach was used: honeybee mortality upon exposure was initially tested in cage, and the colonies development monitored. Morphological and ultrastructural analyses via light and transmission electron microscopy were carried out on the gut of larvae and forager honeybees. Moreover, the main pollen foraging sources and the fungal gut microbiota were studied using Next Generation Sequencing; the gut core bacterial taxa were quantified via qPCR. The mortality test showed a negative effect on honeybee survival when exposed to agrochemicals and their mixture in cage but not confirmed at colony level. Microscopy analyses on the gut epithelium indicated no appreciable morphological changes in larvae, newly emerged and forager honeybees exposed in field to the agrochemicals. Nevertheless, the gut microbial profile showed a reduction of Bombilactobacillus and an increase of Lactobacillus and total fungi upon mixture application. Finally, we highlighted for the first time a significant honeybee diet change after pesticide exposure: penconazole, alone or in mixture, significantly altered the pollen foraging preference, with honeybees preferring Hedera pollen. Overall, our in-hive results showed no severe effects upon administration of sublethal doses of thiacloprid and penconazole but indicate a change in honeybees foraging preference. A possible explanation can be that the different nutritional profile of the pollen may offer better recovery chances to honeybees.

The Cuticular Hydrocarbons of Dasineura Oleae Show Differences Between Sex, Adult Age and Mating Status.

In insects, cuticular lipids prevent water loss and act as semiochemicals. Because of their ecological function, the profile change across the insects' sex and development offers insight into insect biology and possible tools for pest management. Here, the first work on cecidomyiid cuticular extracts is proposed considering Dasineura oleae (Diptera: Cecidomyiidae) males and females at different adult ages (0-12 h, 12-24 h, 24-36 h) and distinct sexual conditions (virgin and mated). A set of 49 compounds were recorded (12 alkanes, 1 monomethyl alkane, 11 fatty acids, 4 esters, 1 aldehyde, 1 allylbenzene, 1 amine, 1 flavonoid, 1 ketone, 1 phenol, 1 steradiene, 1 sterol, 1 terpene, 1 triterpene and 11 unknown compounds), and 18 of them showed significant differences between groups. Among alkanes, hexacosane (nC26) exhibited a decreasing trend from the youngest to the oldest females, while pentacosane (nC25) and nonacosane (nC29) showed a decreasing trend from 0 to 12 h to 12-24 h virgin females. In addition, nonadecane (nC19) was significantly more abundant in the youngest males compared to older males and females. The alkanes nC25, nC26 and nC29 have been reported to be age-related also in other dipterans, while nC19 has been described as gender-specific chemical cue for platygastrid parasitoids. Further behavioural trials and analyses are required to assign the specific ecological roles to the characterized compounds. Our results may contribute to develop new low-impact control strategies relying on the manipulation of D. oleae's chemical communication (e.g. disruption of mating or species recognition). HIGHLIGHTS: • Cuticular hydrocarbons are often involved in dipteran intraspecific communication. • We explored the cuticular profile of D. oleae at different age, sex, mating condition. • Five alkanes and one mono-methyl alkane showed differences among groups. • Linoleic acid is the most abundant compound in virgins, absent in mated insects. • Eleven compounds disappear in mated insects, but were present in all virgins.

Infestation of the gall midge Dasineura oleae provides first evidence of induced plant volatiles in olive leaves.

In this study, we present the first characterization of herbivore-induced plant volatiles (HIPVs) released from infested olive leaves. The gall midge Dasineura oleae is a specific pest of Olea europaea and endemic of the Mediterranean Basin, an area in which severe outbreaks currently occurred. Little is known about the damage caused by the pest and the relationship with its host. Since gall formation and larval feeding activity may lead to the release of specific plant volatile compounds, we investigated the volatile profiles emitted from infested plants compared with healthy plants under both laboratory and field conditions. Additionally, the volatiles emitted from mechanically damaged plants were considered. A blend of 12 volatiles was emitted from olive trees infested by D. oleae. Of these, ß-copaene, ß-ocimene, cosmene, unknown 1 and unknown 3 were found to be exclusively emitted in infested plants. The emission of germacrene-D, (E,E)-α-farnesene, and (Z,E)-α-farnesene, α-copaene, (E)-4,8-dimethylnona-1,3,7-triene, (E)-ß-guaiene and heptadecane significantly increased in infested trees. Linalool, ß-copaen-4-α-ol, ß-bourbonene, ß-cubebene, ß-elemene, ß-copaene and δ-amorphene were found only in the field trial and showed differences depending on the level of infestation and the plant stage. (Z)-3-Hexenol, (E)-4-oxohen-2-enal, and 2-(2-butoxyethoxy)-ethanol, were exclusively emitted from the leaves after mechanical damage. In a field trial in Italy, we also demonstrated spring synchronization between adults of D. oleae and O. europaea trees. Analyses of morphoanatomical malformations of gall leaves showed that tissue alterations occur at the spongy parenchyma causing an increase of the leaf blade thickness. We speculate that tissue alterations may lead to HIPV release, in turn potentially attracting D. oleae natural enemies.

Impact of Chronic Exposure to Two Neonicotinoids on Honey Bee Antennal Responses to Flower Volatiles and Pheromonal Compounds.

Volatile compounds provide important olfactory cues for honey bees (Apis mellifera L.), which are essential for their ecology, behavior, and social communication. In the external environment bees locate food sources by the use of floral scents, while inside the hive, pheromones such as the queen mandibular pheromone (QMP) and alarm pheromones serve important functions in regulating colony life and inducing aggressive responses against intruders and parasites. Widely reported alterations of various behaviors in- and outside the hive following exposure to pesticides could therefore be associated with a disturbance of odor sensitivity. In the present study, we tested the effects of neonicotinoid pesticides at field concentrations on the ability of honey bees to perceive volatiles at the very periphery of the olfactory system. Bee colonies were subjected to treatments during the summer with either Imidacloprid or Thiacloprid at sublethal concentrations. Antennal responses to apple (Malus domestica L.) flower volatiles were studied by GC-coupled electro-antennographic detection (GC-EAD), and a range of volatiles, a substitute of the QMP, and the alarm pheromone 2-heptanone were tested by electroantennography (EAG). Short-term and long-term effects of the neonicotinoid treatments were investigated on bees collected in the autumn and again in the following spring. Treatment with Thiacloprid induced changes in antennal responses to specific flower VOCs, with differing short- and long-term effects. In the short term, increased antennal responses were observed for benzyl-alcohol and 1-hexanol, which are common flower volatiles but also constituents of the honey bee sting gland secretions. The treatment with Thiacloprid also affected antennal responses to the QMP and the mandibular alarm pheromone 2-heptanone. In the short term, a faster signal degeneration of the response signal to the positive control citral was recorded in the antennae of bees exposed to Thiacloprid or Imidacloprid. Finally, we observed season-related differences in the antennal responses to multiple VOCs. Altogether, our results suggest that volatile-specific alterations of antennal responses may contribute to explaining several behavioral changes previously observed in neonicotinoid-exposed bees. Treatment effects were generally more prominent in the short term, suggesting that adverse effects of neonicotinoid exposure may not persist across generations.

Remote monitoring of Cydia pomonella adults among an assemblage of nontargets in sex pheromone-kairomone-baited smart traps.

BACKGROUND: Captures of codling moth, Cydia pomonella (L.), in traps are used to establish action thresholds and time insecticide sprays. The need for frequent trap inspections in often remote orchards has created a niche for remote sensing smart traps. A smart trap baited with a five-component pheromone-kairomone blend was evaluated for codling moth monitoring among an assemblage of other nontargets in apple and pear orchards. RESULTS: Codling moth captures did not differ between the smart trap and a standard trap when both were checked manually. However, the correlation between automatic and manual counts of codling moth in the smart traps was low, R2  = 0.66 ÷ 0.87. False-negative identifications by the smart trap were infrequent <5%, but false-positive identifications accounted for up to 67% of the count. These errors were primarily due to the misidentification of three moth species of fairly similar-size to codling moth: apple clearwing moth Synanthedon myopaeformis (Borkhausen), oriental fruit moth Grapholita molesta (Busck), and carnation tortrix Cacoecimorpha pronubana (Hübner). Other false-positive counts were less frequent and included the misidentifications of dipterans, other arthropods, patches of moth scales, and the double counting of some moths. CONCLUSION: Codling moth was successfully monitored remotely with a smart trap baited with a nonselective sex pheromone-kairomone lure, but automatic counts were inflated in some orchards due to mischaracterizations of primarily similar-sized nontarget moths. Improved image-identification algorithms are needed for smart traps baited with less-selective lures and with lure sets targeting multiple species.

Comparison of New Kairomone-Based Lures for Cydia pomonella (Lepidoptera: Tortricidae) in Italy and USA.

Studies were conducted during the period 2019/2020 to evaluate the effectiveness of four lures for codling moth (Cydia pomonella L.) in pome fruits in Italy and the USA. Multi-component blends of sex pheromone ((E,E)-8,10-dodecadien-1-ol, PH), pear ester ((E,Z)-2,4-ethyl decadienoate, PE), (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and pyranoid linalool oxide (6-ethenyl-2,2,6-trimethyloxan-3-ol, LOX) were loaded in either a halobutyl elastomer septum or a PVC matrix and always used in combination with acetic acid (AA) loaded in a closed membrane co-lure. Total moth capture was significantly greater with the PVC than the septum lure loaded with PH/PE + AA in both countries. Female capture in the USA study was significantly greater for 8 weeks in traps baited with the PE/DMNT/LOX blend + AA co-lure than with other lures and adding PH to this blend in a PVC lure significantly reduced female capture. In contrast, female capture in Italy did not differ among lures and counts were similar in both apple and pear crops treated with or without mating disruption. These results suggest that the effectiveness of 'female removal' strategies to manage codling moth may be geographically limited and further comparisons are needed in other production regions and in walnut.

Botanical Origin of Pesticide Residues in Pollen Loads Collected by Honeybees During and After Apple Bloom.

Honeybees closely rely on insect-pollinated plants for their survival. Each forager bee displays a tendency of loyalty toward specific plant species during the many daily foraging flights. Due to the ease of collection, pollen loads have been extensively used as a proxy for detection of pesticide residues. Pollen is the main protein food source for colonies, and its contamination has also been addressed as a reason for the colony losses phenomenon. As honeybees fly over a variable but wide range territory, they might collect pollen from both agricultural, urban and wild environments, also displaying considerable preferences in botanical sources between colonies of the same apiary. It is thus difficult to address the source of the pesticide contamination, when pollen is analyzed as a whole. In the current study, a practical and reliable approach has been proposed to narrow down the source of contamination. Pollen loads have been collected from colonies placed in eight locations over large apple orchard extensions in Trentino-South Tyrol region (Italy), during and 2 weeks after apple blossom. The pollen loads have been separated by the color due to the predominant plant species. On each color group, palynology and multi-residual chemical analyses have been performed in parallel. The pollen hazard quotient (PHQ) was used to estimate the risk to honeybees of each color group and of the total collected pollen. Apple and dandelion pollen were the main portions of the first collection, while a greater variety emerged after the apple blossom. Dandelion was always present in the samples. The frequency and the amount of pesticide residues differed according to the collection periods, the locations and the pollen color groups. The amount of insecticide residues increased after the apple blossom, while no difference between the period was found on fungicide residues. The PHQ values were higher after the blossom due to the insecticide contribution, with highest values of 160,000 and 150,000. The variations within samples did not allow to identify a unique source of contamination, whereas it seems that the pollen from plants outside the agricultural areas has as much residues as the pollen from apple orchards.

Tree colonization by the Asian longhorn beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae): effect of habitat and tree suitability.

Tree colonization and feeding activity of the invasive wood-borer Asian longhorn beetle (Anoplophora glabripennis), an Asian pest introduced into North America and Europe, was studied in a newly invaded area in Italy. The hypothesis being tested was that the reproductive success of the insect depend on habitat type and tree suitability. Adult beetles were caged on branches of host and nonhost species, in both urban and forest habitats. Two months later, number and size of feeding patches on plant tissues, eggs laid, and surviving larvae were assessed. Bark concentration of C and N was also measured from the same trees. Results indicated that the mean area of plant tissues consumed by adult feeding was significantly larger on trees growing in forest than in urban habitat, although within the same habitat there were no differences between susceptible and nonsusceptible trees. ALB tree colonization, in terms of number of eggs laid and young larvae survival, was not affected by habitat while it was higher on susceptible trees. Although trees growing in forests had a lower nitrogen concentration, they allowed colonization rates similar to those of trees growing in the urban habitat. Hence, the amount of carbon and nitrogen did not fully explain tree suitability or habitat selection. We suggest compensatory feeding as a potential mechanism that might explain this peculiar situation, as supported by a more intensive feeding activity recorded on trees in the forest. Suitability of different trees may be due to other factors, such as secondary chemical compounds.