Characterization of Olive Fruit Damage Induced by Invasive Halyomorpha halys.

The brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive species causing economic crop losses. This species was recently detected attacking olive fruits. The aim of this study was to characterize feeding damage. Olive samples were initially collected from a field where H. halys was reported to cause damage to olive fruits. Hence, we conducted a field trial on the Moraiolo variety using sleeve cages to test the effect of H. halys feeding pressure on olive fruit drop and evaluated the effect of feeding on fruit quality. We tested two densities of H. halys (two or eight adults/cage) at two different stages of olive development, pre- and post-pit hardening. High pressure of H. halys before pit hardening caused a significant fruit drop compared to the control. In addition, chemical analysis of damaged and infested fruits revealed higher levels of total phenols compared to healthy fruits. These findings indicate that feeding by H. halys induced a stress response in the plants that could translate in quality variations in the olive drupes.

Morpho-functional Analysis of the Head Glands in Three Auchenorrhyncha Species and Their Possible Biological Significance.

The Cicadomorpha Philaenus spumarius, Neophilaenus campestris, and Cicadella viridis are known transmitters of the bacterium Xylella fastidiosa. Here, we studied the ultrastructural organization of their cephalic glands. Our investigations with scanning, transmission, focused ion beam-scanning electron microscopes and light microscope revealed for the first time in Auchenorrhyncha the presence of two types of cephalic glands. Both belonged to the Class III epidermal glands, according to the Noirot and Quennedey classification. Type A glands were the most common, being mainly located around antennae, lorum, and gena. Moreover, these glands were observed also on the abdomen and thorax, always in association with sensilla trichoidea. The second type of glands (type B) were located exclusively at the apical part of the postclypeus in P. spumarius and N. campestris. The ultrastructural organization was similar in both types, being composed of a secretory cell and a conducting canal. Differences were observed in the width of the cuticular opening, being smaller in the type II glands. In addition, we have recorded the presence of a maxillary sensory pit in all species and described sensilla trichoidea ultrastructural organization. Finally, we discussed the ultrastructural organization of the glands and their potential biological role.

Improved Captures of the Invasive Brown Marmorated Stink Bug, Halyomorpha halys, Using a Novel Multimodal Trap.

Capture strategies for the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), are challenging. Here we developed and evaluated a multimodal trap which combines visual and olfactory stimuli. Visual stimuli consisted of LEDs emitting UV-A and visible light. Olfactory stimuli were comprised of the synthetic aggregation pheromone and odours from trapped H. halys individuals. Stink bug attraction at different wavelengths was evaluated in laboratory two-choice bioassays, and different prototypes of the trap were tested in 2021 in natural, agricultural, and urban settings. Traps with a combination of UV-A and blue or green visible wavelengths provided higher H. halys attraction (up to ~8-fold) compared to traditional sticky or small pyramidal traps. The concurrent presence of synthetic pheromone and LED had a synergistic effect on H. halys positive phototaxis. Further development and implementation of the multimodal trap is discussed for prospective use in attract-and-kill or push-pull strategies.

Behavioural and electrophysiological responses of Philaenus spumarius to odours from conspecifics.

The meadow spittlebug, Philaenus spumarius L. (Hemiptera: Auchenorrhyncha: Aphrophoridae), is the main vector of Xylella fastidiosa subsp. pauca strain ST53, the causal agent of the Olive Quick Decline Syndrome. Philaenus spumarius and other Auchenorrhyncha are known to communicate via vibrations, whereas the possible occurrence of semiochemical communication has been poorly investigated so far. Through a chemical ecology approach, we provide evidence of intraspecific chemical communication in P. spumarius. In Y-tube olfactometer bioassays, males were attracted to unmated females as well as toward the headspace volatile extracts collected from unmated females. Conversely, females did not respond to unmated male volatiles or their extracts, nor did males and females respond to volatiles from individuals of the same sex. Electroantennography assays of unmated male and female headspace extracts elicited measurable responses in the antennae of both sexes. Male responses to body wash extracts from both sexes were stronger compared to female responses. Thus, suggesting the presence of compounds that are highly detected by the male's olfactory system. The female head seemed to be the source of such compounds. This is the first record of intraspecific chemical communication in P. spumarius and one of the very few records in Auchenorrhyncha. Possible biological roles are under investigation.

Field and Laboratory Efficacy of Low-Impact Commercial Products in Preventing Olive Fruit Fly, Bactrocera oleae, Infestation.

The olive fruit fly, Bactrocera oleae, is the key pest of olive trees in several areas of the world. Given the need for the development of sustainable control methods, preventive tools, based on the manipulation of pest behaviour, must be considered. Here, under field and laboratory conditions, we tested the efficacy of different products in preventing B. oleae infestation. A field trial was conducted, from July to November 2020, in an olive orchard located in Central Italy. A table olive variety was selected and sprayed with rock powder, propolis, the mixture of both, copper oxychloride, or water (control). All treatments, except propolis, caused a reduction of B. oleae oviposition in olives, compared to the control. The mixture allowed the strongest reduction of fly infestation throughout the season, suggesting a synergistic effect. Behavioural no-choice assays were conducted to better understand the effects of treatments on B. oleae females. Compared to the control, females showed a lower preference for the central area of an arena containing an olive twig bearing two olive fruits, fully developed, but still green, treated with rock powder, plus propolis mixture. For all treatments, B. oleae showed lower oviposition events, suggesting deterrence to oviposition. Our results indicate that the tested products may have value against B. oleae, within integrated pest management (IPM) and organic agriculture.

Olfactory responses of Trissolcus mitsukurii to plants attacked by target and non-target stink bugs suggest low risk for biological control.

In crop systems, successful management of invasive insect herbivores can be achieved through the introduction of exotic biocontrol agents, parasitoids or predators, having a coevolutionary history with the pest. To avert threats to local biodiversity, recent legislations require a risk assessment for the organism to be released. Evaluation of its ability to exploit, for host location, odours associated with target and non-target species is crucial for a better definition of its ecological host range. Using Y-tube olfactometer bioassays in a quarantine laboratory, we investigated the ability of the Asian egg parasitoid Trissolcus mitsukurii (Hymenoptera: Scelionidae) to exploit odours associated with the global invader Halyomorpha halys (Hemiptera: Pentatomidae) and with non-target stink bugs native to Southern Europe. We demonstrated that T. mitsukurii is attracted by plants exposed to feeding and egg deposition of the coevolved H. halys and the native Nezara viridula, while it is not attracted by physogastric (gravid) females or eggs alone. Remarkably, T. mitsukurii is repelled by plants bearing eggs of the beneficial Arma custos. Our results contribute to a more thorough and nuanced assessment of the potential non-target risks in the case of mass-release of parasitoids as part of a biological control programme for invasive stink bugs.

A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the "tip of the iceberg" since thousands of sequences remain uncharacterized in this enzyme family alone.

Stage-specific expression of an odorant receptor underlies olfactory behavioral plasticity in Spodoptera littoralis larvae.

BACKGROUND: The detection of environmental cues and signals via the sensory system directs behavioral choices in diverse organisms. Insect larvae rely on input from the chemosensory system, mainly olfaction, for locating food sources. In several lepidopteran species, foraging behavior and food preferences change across larval instars; however, the molecular mechanisms underlying such behavioral plasticity during larval development are not fully understood. Here, we hypothesize that expression patterns of odorant receptors (ORs) change during development, as a possible mechanism influencing instar-specific olfactory-guided behavior and food preferences. RESULTS: We investigated the expression patterns of ORs in larvae of the cotton leafworm Spodoptera littoralis between the first and fourth instar and revealed that some of the ORs show instar-specific expression. We functionally characterized one OR expressed in the first instar, SlitOR40, as responding to the plant volatile, ß-caryophyllene and its isomer α-humulene. In agreement with the proposed hypothesis, we showed that first but not fourth instar larvae responded behaviorally to ß-caryophyllene and α-humulene. Moreover, knocking out this odorant receptor via CRISPR-Cas9, we confirmed that instar-specific responses towards its cognate ligands rely on the expression of SlitOR40. CONCLUSION: Our results provide evidence that larvae of S. littoralis change their peripheral olfactory system during development. Furthermore, our data demonstrate an unprecedented instar-specific behavioral plasticity mediated by an OR, and knocking out this OR disrupts larval behavioral plasticity. The ecological relevance of such behavioral plasticity for S. littoralis remains to be elucidated, but our results demonstrate an olfactory mechanism underlying this plasticity in foraging behavior during larval development.

Effect of microplastics and watering regimes on a plant-soil system: Data on behavioural responses of an insect herbivore.

The data presented here are related to the article titled "Microplastics alter behavioural responses of an insect herbivore to a plant-soil system" by Rondoni, G., Chierici, E., Agnelli, A., Conti, E. (2021). The data describe the changes in the attractiveness of a plant-soil system towards females of a herbivorous fungus gnat (Diptera: Sciaridae) when exposed to different combinations of the following treatments: (1) low or high plant (lentil)-soil watering regime; (2) absence (0%) or presence (5%) of HDPE microplastics in soil; (3) 1-day or 7-day duration of HDPE presence; (4) addition of fungus mycelium to the plant-soil system. We report data of female behaviour, i.e. the residence time in choice vs. no-choice sector of one-way olfactometers using a multiple olfactometer device.

Antennal Transcriptome Analysis and Identification of Candidate Chemosensory Genes of the Harlequin Ladybird Beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae).

In predatory ladybirds (Coleoptera: Coccinellidae), antennae are important for chemosensory reception used during food and mate location, and for finding a suitable oviposition habitat. Based on NextSeq 550 Illumina sequencing, we assembled the antennal transcriptome of mated Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) males and females and described the first chemosensory gene repertoire expressed in this species. We annotated candidate chemosensory sequences encoding 26 odorant receptors (including the coreceptor, Orco), 17 gustatory receptors, 27 ionotropic receptors, 31 odorant-binding proteins, 12 chemosensory proteins, and 4 sensory neuron membrane proteins. Maximum-likelihood phylogenetic analyses allowed to assign candidate H. axyridis chemosensory genes to previously described groups in each of these families. Differential expression analysis between males and females revealed low variability between sexes, possibly reflecting the known absence of relevant sexual dimorphism in the structure of the antennae and in the distribution and abundance of the sensilla. However, we revealed significant differences in expression of three chemosensory genes, namely two male-biased odorant-binding proteins and one male-biased odorant receptor, suggesting their possible involvement in pheromone detection. Our data pave the way for improving the understanding of the molecular basis of chemosensory reception in Coccinellidae.

Learning can be detrimental for a parasitic wasp.

Animals have evolved the capacity to learn, and the conventional view is that learning allows individuals to improve foraging decisions. The parasitoid Telenomus podisi has been shown to parasitize eggs of the exotic stink bug Halyomorpha halys at the same rate as eggs of its coevolved host, Podisus maculiventris, but the parasitoid cannot complete its development in the exotic species. We hypothesized that T. podisi learns to exploit cues from this non-coevolved species, thereby increasing unsuccessful parasitism rates. We conducted bioassays to compare the responses of naïve vs. experienced parasitoids on chemical footprints left by one of the two host species. Both naïve and experienced females showed a higher response to footprints of P. maculiventris than of H. halys. Furthermore, parasitoids that gained an experience on H. halys significantly increased their residence time within the arena and the frequency of re-encounter with the area contaminated by chemical cues. Hence, our study describes detrimental learning where a parasitoid learns to associate chemical cues from an unsuitable host, potentially re-enforcing a reproductive cul-de-sac (evolutionary trap). Maladaptive learning in the T. podisi-H. halys association could have consequences for population dynamics of sympatric native and exotic host species.

Data on soil physicochemical properties and biodiversity from conventional, organic and organic mulch-based cropping systems.

The data presented here are related to the article entitled "Soil functions are affected by transition from conventional to organic mulch-based cropping system"[1]. Data were collected in 2016 in a processing tomato field located near Perugia, Italy. In details, data were collected in three differently managed processing tomato cropping systems: conventional integrated (INT); traditional organic with cover crops and conventional tillage (ORG); and organic coupled with conservation agriculture, with mulch-based cover crop and no-tillage (ORG+). We report data on the impact of each cropping system on crop biomass and yield, soil physicochemical properties, size and structure of soil microbial community, soil invertebrate biodiversity and habitat provision (predator-prey trophic interactions).

An Egg Parasitoid Efficiently Exploits Cues From a Coevolved Host But Not Those From a Novel Host.

Egg parasitoids have evolved adaptations to exploit host-associated cues, especially oviposition-induced plant volatiles and odors of gravid females, when foraging for hosts. The entire host selection process is critical for successful parasitism and relevant in defining host specificity of parasitoids. We hypothesized that naïve egg parasitoid females reared on their coevolved host are able to exploit cues related to the coevolved host but not those from a novel host. We used the egg parasitoid Trissolcus japonicus, its coevolved host Halyomorpha halys, and the non-coevolved host Podisus maculiventris to evaluate this hypothesis. H. halys, a polyphagous pest native from Eastern Asia, has invaded North America and Europe, resulting in serious damage to crops. T. japonicus is the most effective egg parasitoid of H. halys in its native area and thus considered a major candidate for biological control. This parasitoid was detected in North America and Europe as a result of accidental introductions. Laboratory host range of T. japonicus includes P. maculiventris, an American predatory stink bug used as a biological control agent of several pests. Using T. japonicus reared on its natural host H. halys, we tested in a Y-tube olfactometer the responses of naïve parasitoid females to volatiles from tomato plants with a deposited egg mass and feeding punctures of either H. halys or P. maculiventris. Additionally, using two different olfactometer set-ups, we tested T. japonicus responses to volatiles emitted by eggs and mature males and females of H. halys or P. maculiventris. Tomato plants subjected to oviposition and feeding by H. halys were preferred by the wasp compared to clean plants, suggesting a possible activation of an indirect defense mechanism. Furthermore, T. japonicus females were attracted by cues from gravid females and mature males of H. halys but not from eggs. By contrast, naïve parasitoid females never responded to cues associated with P. maculiventris, although this non-target host is suitable for complete parasitoid development. Such lack of responses might reduce the probability of T. japonicus locating and parasitizing P. maculiventris under field conditions. Our experimental approach properly simulates the parasitoid host-location process and could be combined with the required host specificity tests for risk assessment in biological control programs.

Mating Status of an Herbivorous Stink Bug Female Affects the Emission of Oviposition-Induced Plant Volatiles Exploited by an Egg Parasitoid.

Insect parasitoids are under selection pressure to optimize their host location strategy in order to maximize fitness. In parasitoid species that develop on host eggs, one of these strategies consists in the exploitation of oviposition-induced plant volatiles (OIPVs), specific blends of volatile organic compounds released by plants in response to egg deposition by herbivorous insects. Plants can recognize insect oviposition via elicitors that trigger OIPVs, but very few elicitors have been characterized so far. In particular, the source and the nature of the elicitor responsible of egg parasitoid recruitment in the case of plants induced with oviposition by stink bugs are still unknown. In this paper, we conducted behavioral and molecular investigations to localize the source of the elicitor that attracts egg parasitoids and elucidate the role of host mating in elicitation of plant responses. We used as organism study model a tritrophic system consisting of the egg parasitoid Trissolcus basalis, the stink bug host Nezara viridula and the plant Vicia faba. We found that egg parasitoid attraction to plant volatiles is triggered by extracts coming from the dilated portion of the stink bug spermathecal complex. However, attraction only occurs if extracts are obtained from mated females but not from virgin ones. Egg parasitoid attraction was not observed when extracts coming from the accessory glands (mesadene and ectadene) of male hosts were applied, either alone or in combination to plants. SDS-PAGE electrophoresis correlated with olfactometer observations as the protein profile of the dilated portion of the spermathecal complex was affected by the stink bug mating status suggesting post-copulatory physiological changes in this reproductive structure. This study contributed to better understanding the host location process by egg parasitoids and laid the basis for the chemical characterization of the elicitor responsible for OIPV emission.

Molecular detection of field predation among larvae of two ladybird beetles is partially predicted from laboratory experiments.

Despite the fact that natural enemies can synergistically contribute to herbivore pest suppression, sometimes predators engage in intraguild predation (IGP) that might dampen trophic cascades. DNA-based gut-content analysis has become common in assessing trophic connections and biocontrol potential by predators in field systems. Here, we developed a molecular technique that can be used to unravel predation among two ladybirds, Coccinella septempunctata and Hippodamia variegata, and their shared prey, Aphis gossypii. Both ladybirds may provide effective control of the pest. Therefore, understanding their likelihood to engage in IGP is crucial for conservation biological control. Ladybird specimens were collected in melon crop. DNA extraction, primer design and evaluation were conducted. Detectability of prey DNA did not differ significantly between the two ladybirds. H. variegata exhibited higher predation on A. gossypii than C. septempunctata (90.6% vs. 70.9%) and data correction based on DNA detectability confirmed this ranking. IGP was similar among the two species, although corrected data might suggest a stronger predation by C. septempunctata. Intriguingly, IGP by C. septempunctata was lower than predicted by laboratory bioassays, possibly due to the high complexity that arises under field conditions. Implications of our results for biological control and perspectives for ecological network analysis are discussed.

An invasive insect herbivore disrupts plant volatile-mediated tritrophic signalling.

Plants respond to insect attack by emission of volatile organic compounds, which recruit natural enemies of the attacking herbivore, constituting an indirect plant defence strategy. In this context, the egg parasitoid Trissolcus basalis is attracted by oviposition-induced plant volatiles emitted by Vicia faba plants as a consequence of feeding and oviposition by the pentatomid host Nezara viridula. However, this local tritrophic web could be affected by the recent invasion by the alien pentatomid bug Halyomorpha halys, an herbivore that shares the same environments as native pentatomid pests. Therefore, we investigated in laboratory conditions the possible impact of H. halys on the plant volatile-mediated signalling in the local tritrophic web V. faba-N. viridula-T. basalis. We found that T. basalis wasps were not attracted by volatiles induced in the plants by feeding and oviposition activities of H. halys, indicating specificity in the wasps' response. However, the parasitoid attraction towards plant volatiles emitted as a consequence of feeding and oviposition by the associated host was disrupted when host, N. viridula, and non-associated host, H. halys, were concurrently present on the same plant, indicating that invasion by the alien herbivore interferes with established semiochemical webs. These outcomes are discussed in a context of multiple herbivory by evaluating the possible influences of alien insects on local parasitoid foraging behaviour.

Behavioural and physiological responses to prey-related cues reflect higher competitiveness of invasive vs. native ladybirds.

Understanding the traits that might be linked with biological invasions represents a great challenge for preventing non-target effects on local biodiversity. In predatory insects, the ability to exploit habitats for oviposition and the physiological response to prey availability differs between species. Those species that respond more readily to environmental changes may confer to their offspring a competitive advantage over other species. Here, we tested the hypothesis that the invasive Harmonia axyridis (Coleoptera: Coccinellidae) makes better use of information from a plant-prey (Vicia faba - Aphis fabae) system compared to the native Oenopia conglobata. Y-tube olfactometer bioassays revealed that both species used olfactory cues from the system, but H. axyridis exhibited a more complete response. This species was also attracted by plants previously infested by aphids, indicating the capacity to exploit volatile synomones induced in plants by aphid attack. Oocyte resorption was investigated when different olfactory stimuli were provided under prey shortage and the readiness of new oogenesis was measured when prey was available again. H. axyridis exhibited higher plasticity in oogenesis related to the presence/absence of plant-aphid volatiles. Our results support the hypothesis that H. axyridis is more reactive than O. conglobata to olfactory cues from the plant-prey system.

Foraging behaviour of an egg parasitoid exploiting plant volatiles induced by pentatomids: the role of adaxial and abaxial leaf surfaces.

Several phases of herbivorous insect attack including feeding and oviposition are known to induce plant defenses. Plants emit volatiles induced by herbivores to recruit insect parasitoids as an indirect defense strategy. So far, volatiles induced by herbivore walking and their putative role in the foraging behavior of egg parasitoids have not been investigated. In this paper we studied the response of the egg parasitoid Trissolcus basalis toward volatiles emitted by Vicia faba plants as consequence of the walking activity of the host Nezara viridula. Olfactometer bioassays were carried out to evaluate wasp responses to plants in which the abaxial or the adaxial surfaces were subjected to walking or/and oviposition. Results showed that host female walking on the abaxial but not on the adaxial surface caused a repellence effect in T. basalis 24 h after plant treatment. The emission of active volatiles also occurred when the leaf was turned upside-down, indicating a specificity of stress localization. This specificity was supported by the results, which showed that oviposition combined with feeding elicit the induction of plant volatiles, attracting the parasitoid, when the attack occurred on the abaxial surface. Analyses of plant volatile blends showed significant differences between the treatments.

The role of contact chemoreception in the host location process of an egg parasitoid.

Taste allows insects to detect palatable or toxic foods, identify a mate, and select appropriate oviposition sites. The gustatory system strongly contributes to the survival and reproductive success of many species, yet it is rarely studied in insect parasitoids. In order to locate and assess a host in which they will lay their eggs, female wasps actively search for chemical cues using their sensory organs present mainly on the antennae. In this paper, we studied the role of antennal taste sensilla chaetica in the perception of contact semiochemicals in Trissolcus brochymenae (Hymenoptera: Platygastridae), an egg parasitoid of the brassicaceae pest Murgantia histrionica (Heteroptera: Pentatomidae). Methanolic extracts obtained from male and female hosts elicited action potentials in taste neurons housed in antennal sensilla chaetica, indicating that these sensilla are involved in the perception of non volatile host kairomones. In behavioural assays, wasp females displayed an intense searching behaviour in open arenas treated with host extracts, thus confirming that these kairomones are soluble in polar solvents. We further investigated the extracts by Gas Chromatography-Mass Spectrometry (GC-MS) and found that they contain several compounds which are good candidates for these contact kairomones. This study contributes to better understanding contact chemoreception in egg parasitoids and identifying gustatory receptor neurons involved in the host location process.

The response of an egg parasitoid to substrate-borne semiochemicals is affected by previous experience.

Animals can adjust their behaviour according to previous experience gained during foraging. In parasitoids, experience plays a key role in host location, a hierarchical process in which air-borne and substrate-borne semiochemicals are used to find hosts. In nature, chemical traces deposited by herbivore hosts when walking on the plant are adsorbed by leaf surfaces and perceived as substrate-borne semiochemicals by parasitoids. Chemical traces left on cabbage leaves by adults of the harlequin bug (Murgantia histrionica) induce an innate arrestment response in the egg parasitoid Trissolcus brochymenae characterized by an intense searching behaviour on host-contaminated areas. Here we investigated whether the T. brochymenae response to host walking traces left on leaf surfaces is affected by previous experience in the context of parasitoid foraging behaviour. We found that: 1) an unrewarded experience (successive encounters with host-contaminated areas without successful oviposition) decreased the intensity of the parasitoid response; 2) a rewarded experience (successful oviposition) acted as a reinforcing stimulus; 3) the elapsed time between two consecutive unrewarded events affected the parasitoid response in a host-gender specific manner. The ecological role of these results to the host location process of egg parasitoids is discussed.