What's on the menu? A novel molecular gut content analysis to investigate the feeding behavior of phytophagous insects.

The relationship between phytophagous insects and plants is a central aspect of food webs and ecosystem functioning. The introduction of new species into an environment can have significant impacts on the food web of a native ecosystem. In many cases, there is a lack of knowledge on the biology and feeding behavior of invasive species prior their introduction and in the invaded regions. Gut content analyses of insects have provided valuable information on the host spectrum of insects. However, current approaches are time-consuming and costly. Here, we describe a new molecular gut content analysis (GCA) approach using the Oxford Nanopore (ONT) Flongle sequencing platform to characterize the plant DNA present in the gut of the highly polyphagous insect species Halyomorpha halys. We demonstrate that this technique efficiently amplifies and correctly identifies plant DNA in a mock community. We performed a feeding experiment to determine the sensitivity of this approach and to assess how long the plant DNA can be detected. All plants used in the feeding experiment were correctly identified and detected after 56 days. Surprisingly, we also detected various plant genera that were not included in the feeding experiment and thus were likely ingested months before the experiment. Our study suggests that the GCA using the ONT Flongle sequencing platform represents a rapid and cost-efficient diagnosis of the dietary preferences, host range, and the diversity of consumed plant species of pest insects with high precision.

Evaluation of Various Inoculation Methods on the Effect of Beauveria bassiana on the Plant Growth of Kiwi and on Halyomorpha halys Infestation: A Two-Year Field Study.

In this study, the bioinsecticidal action of a commercial formulation with Beauveria bassiana was evaluated on the new sucking pest in Greece: Halyomorpha halys, of the kiwifruit. Additionally, the biostimulant potential of the same formulation was studied on kiwi growth. The application was performed in three different ways in a commercial field of kiwi crop A. deliciosa "Hayward" field in Arta, Greece: (i) trunk spray, (ii) root injection, and (iii) trunk inoculation. During the 2 years seasons of the experiment, weekly measurements of the H. halys population were determined. The insect is sucking plants nutrients; therefore, the total chlorophyll content in the leaves of the treatments was recorded weekly. In addition, the percentage of infested kiwifruits was estimated at the end of the experiment. Moreover, to study the biostimulant potential of the formulation, growth measurements on stems and leaves were performed during the experiment. Finally, at the kiwi harvest point, the fruit biomass, dimensions, and weight were obtained, and the leaves' proline content was evaluated. The results encourage us to further study this EPF formulation as the bioinsecticidal effect was noted by the reduction in H. halys population, and biostimulant action was perceived by the higher plant biomass.

The multispecies stinkbug iflavirus Halyomorpha halys virus detected in the multispecies stinkbug egg parasitoid microwasp, Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae).

Wasps are important parasitoids of stinkbugs and frequently exposed to various types of microorganisms through environmental contact and fecal-oral transmission route. Many parasitize stinkbug eggs and are commercially used in the field to control insect population. The parasitoid T. podisi is known for its high parasitism capacity and ability to target multiple species of stinkbugs. In this study we asked whether T. podisi exposed to eggs infected by a multispecies asymptomatic stinkbug virus, the Halyomorpha halys virus (HhV) would get infected. HhV is a geographically distributed multispecies iflavirus previously found to infect four stinkbug hosts, including three Brazilian species, Chinavia ubica, Euschistus heros and Diceraeus melacanthus, and T. posidi can parasitize all of them. As results, RT-PCR screening revealed positive samples for the HhV genome in two out of four tested pools of T. podisi, whereas the antigenome, indicative of replicative activity, was not detected. The wasps were raised in E. heros eggs that presented both the genome and the antigenome forms of the HhV genome. Subsequent RNA-deep sequencing of HhV positive T. podisi RNA pools yielded a complete genome of HhV with high coverage. Phylogenetic analysis positioned the isolate HhV-Tp (isolate Telenomus podisi) alongside with the stinkbug HhV. Analysis of transcriptomes from several hymenopteran species revealed HhV-Tp reads in four species. However, the transmission mechanism and the ecological significance of HhV remain elusive, warranting further studies to illuminate both the transmission process and its capacity for environmental propagation using T. podisi as a potential vector.

Distribution and prevalence of viral genomes in Italian populations of the invasive brown marmorated stink bug Halyomorpha halys.

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

Combining Irradiation and Biological Control against Brown Marmorated Stink Bug: Are Sterile Eggs a Suitable Substrate for the Egg Parasitoid Trissolcus japonicus?

The brown marmorated stink bug (BMSB), Halyomorpha halys, is a phytophagous invasive pest native to south-eastern Asia, and it is now distributed worldwide. This species is considered to be one of the most damaging insect pests in North America and in Europe. In agriculture, the predominant approach to managing BMSB is based on the use of insecticides, specifically pyrethroids and neonicotinoids. Unfortunately, the biology of the species and its facility to develop mechanisms of resistance to available pesticides has induced farmers and scientists to develop different, least-toxic, and more effective strategies of control. In a territorial area-wide approach, the use of a classical biological control program in combination with other least-toxic strategies has been given prominent consideration. Following exploratory surveys in the native range, attention has focused on Trissolcus japonicus, a small scelionid egg parasitoid wasp that is able to oviposit and complete its larval development in a single egg of H. halys. A common method for detecting egg parasitoids in the native range involves the placement of so-called 'sentinel' egg masses of the pest in the environment for a short period, which are then returned to the laboratory to determine if any of them are parasitized. Outside of the area of origin, the use of fertile sentinel eggs of the alien species may lead to the further release of the pest species; an alternative is to use sterile sentinel eggs to record the presence of new indigenous egg parasitoids or to detect the dispersal of alien species (in this case, T. japonicus) released in a new environment to control the target insect pest species. This study evaluated the performance of three types of sterile sentinel eggs as a suitable substrate for the oviposition and larval development of the egg parasitoid T. japonicus in a context of combining classical biological control with a Sterile Insect Technique (SIT) approach.

Entomopathogens infecting brown marmorated stink bugs before, during, and after overwintering.

The microsporidian, Nosema maddoxi Becnel, Solter, Hajek, Huang, Sanscrainte & Estep, infects brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), populations in North America and Asia and causes decreased fitness in infected insects. This host overwinters as adults, often in aggregations in sheltered locations, and variable levels of mortality occur over the winter. We investigated pathogen prevalence in H. halys adults before, during, and after overwintering. Population level studies resulted in detection of N. maddoxi in H. halys in 6 new US states, but no difference in levels of infection by N. maddoxi in autumn versus the following spring. Halyomorpha halys that self-aggregated for overwintering in shelters deployed in the field were maintained under simulated winter conditions (4°C) for 5 months during the 2021-2022 winter and early spring, resulting in 34.6 ± 4.8% mortality. Over the 2020-2021 and 2021-2022 winters, 13.4 ± 3.5% of surviving H. halys in shelters were infected with N. maddoxi, while N. maddoxi infections were found in 33.4 ± 10.8% of moribund and dead H. halys that accumulated in shelters. A second pathogen, Colletotrichum fioriniae Marcelino & Gouli, not previously reported from H. halys, was found among 46.7 ± 7.8% of the H. halys that died while overwintering, but levels of infection decreased after overwintering. These 2 pathogens occurred as co-infections in 11.1 ± 5.9% of the fungal-infected insects that died while overwintering. Increasing levels of N. maddoxi infection caused epizootics among H. halys reared in greenhouse cages after overwintering.

Releasing and tracking the distribution of adventive Trissolcus japonicus (Hymenoptera: Scelionidae) in Virginia.

Adventive populations of Trissolcus japonicus (Ashmead), an egg parasitoid of the invasive agricultural pest, brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), have been detected in the United States since 2014. Given its importance as an H. halys biocontrol agent, efforts to redistribute T. japonicus began within some US states. Our surveillance for T. japonicus in northwestern Virginia in 2016-2017 yielded annual detections only in 1 county. Thus, to promote its broader establishment, releases of H. halys egg masses parasitized by T. japonicus from Virginia occurred in 2018 (2 releases) and 2020 (1 release) at 9 sites throughout Virginia's tree fruit production regions. Monitoring of T. japonicus and H. halys, using yellow sticky cards deployed in H. halys host trees and pheromone-baited sticky traps, respectively, was conducted from 2018 to 2022. Annual captures of H. halys adults and nymphs appeared to reflect adequate populations to support T. japonicus establishment across most or all sites. Prerelease monitoring yielded a single T. japonicus at 1 site. By 2022, T. japonicus was detected at or near 7 of the remaining 8 release sites, with first detections varying between 1 and 2 yr from the releases in 2018 and 2020. Captures at most sites were very low, but establishment at several locations was indicated by detections in 2-4 seasons. In 2022, T. japonicus surveillance at 11 additional sites in northwestern Virginia yielded detections at all locations, including those at which it had not been detected in 2016-2017, providing evidence for its range expansion.

The Invasive Caucasian Populations of the Brown Marmorated Stink Bug Halyomorpha halys (Hemiptera: Heteroptera: Pentatomidae) Rapidly Adapt Their Ecophysiological Traits to the Local Environmental Conditions.

The ability to rapidly adapt to new environmental conditions is a crucial prerequisite for the wide-scale invasion of pests or intentional introduction of beneficial insects. A photoperiodically induced facultative winter diapause is an important adaptation ensuring synchronization of insect development and reproduction with the local seasonal dynamics of environmental factors. We conducted a laboratory study aimed to compare photoperiodic responses of two invasive Caucasian populations of the brown marmorated stink bug Halyomorpha halys (Hemiptera: Heteroptera: Pentatomidae), which recently invaded neighboring regions with subtropical (Sukhum, Abkhazia) and temperate (Abinsk, Russia) climates. Under the temperature of 25 °C and the near-critical photoperiods of L:D = 15:9 h and 15.5:8.5 h, the population from Abinsk showed a slower pre-adult development and a stronger tendency to enter winter adult (reproductive) diapause compared to the population from Sukhum. This finding agreed with the difference between the local dynamics of the autumnal temperature decrease. Similar adaptive interpopulation differences in the patterns of diapause-inducing responses are known in other insect species but our finding is distinguished by a very short adaptation time: H. halys was first recorded in Sukhum in 2015 and in Abinsk in 2018. Thus, the differences between the compared populations might have evolved over a relatively short span of several years.

Effects of Irradiation on Biology and Mating Behaviour of Wild Males of Brown Marmorated Stink Bug Using a 6 MV Medical Linear Accelerator.

The brown marmorated stink bug, Halyomorpha halys, is a pentatomid bug of Eastern Asian origin that became an economically relevant pest in the Eurasian and American continents. Management of this species is limited to use of chemical insecticides: an inefficient method due to the strong adaptability of the target pest. The sterile insect technique (SIT) is potentially a valid tactic in the search for nontoxic alternatives. In this work, we investigated the suitability of mass-trapped overwintering males, collected during the aggregation phase before the winter diapause, for their release as competitive sterile males in an SIT programme. Differently from previous studies, irradiation was applied with a linear accelerator device that produced high-energy photons. Following a similar scientific protocol with newly emerged irradiated males, the effects of X-ray irradiation on physiological parameters (longevity, fecundity and fertility) were assessed. In addition, behavioural bioassays were carried out in no-choice conditions to evaluate if irradiation interferes with mating processes. The results are very encouraging; the effects of the irradiation at 32 Gy did not differ from the controls in the longevity or fecundity of the exposed overwintering adults. The hatching rate of the eggs laid by the fertile females that had mated with the irradiated males was less than 5%. The results of behavioural bioassays showed that the irradiation did not cause a significant impact on the quality of the sterile males. More research is warranted to evaluate the mating competitiveness of sterile males in semi-field and field conditions.

Performance of two egg parasitoids of brown marmorated stink bug before and after cold storage.

Introduction: The genus Trissolcus includes a number of egg parasitoids that are known to contribute to the control of Halyomorpha halys. The number of progenies, particularly females, is important for the efficient mass rearing of species used in augmentative biological control programs. Cold storage is an important technique for extending the shelf life of natural enemies used in such programs. Methods: We assessed how fecundity, sex ratio, lifespan, and the number of hosts parasitized within 24 h were affected by host density for T. japonicus and T. cultratus when offered fresh H. halys eggs and how these parameters were affected if adult parasitoids were first placed in cold storage (11°C in the dark) for 19 weeks before being used for propagation. Results: The fecundity were 110.2 and 84.2 offspring emerged at 25°C, for parasitoids not placed in cold storage; among the offspring that emerged, 82.6% and 85.6% were female for T. japonicus and T. cultratus, respectively. If first placed in cold storage, T. japonicus and T. cultratus produced 35.1 and 24.6 offspring per female, respectively, although cold storage significantly extended the shelf life. The survival rates of parasitoids that were placed in cold storage were 90.3% and 81.3% for females, and 3.2% and 0.9% for males of T. japonicus and T. cultratus, respectively. The number of hosts parasitized within 24 h was not shown to be density dependent, but it was significantly lower after cold storage. Discussion: This information can be used to estimate the likely production for augmented rearing colonies for use in biological control programs.

Exploration of Candidate Genes Involved in the Biosynthesis, Regulation and Recognition of the Male-Produced Aggregation Pheromone of Halyomorpha halys.

The aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys (Stål), is produced by adult males, and plays an important role in the behavioral regulation of H. halys. However, information on the molecular mechanisms underlying this pheromone's biosynthesis is limited. In this study, HhTPS1, a key candidate synthase gene in the aggregation pheromone biosynthesis pathway of H. halys, was identified. Then, through weighted gene co-expression network analysis, the candidate P450 enzyme genes in the biosynthetic downstream of this pheromone and the related candidate transcription factor in this pathway were also identified. In addition, two olfactory-related genes, HhCSP5 and HhOr85b, involved in the recognition of the aggregation pheromone of H. halys, were detected. We further identified the key amino acid sites of HhTPS1 and HhCSP5 that interact with substrates by using molecular docking analysis. This study provides basic information for further investigations into the biosynthesis pathways and recognition mechanisms of aggregation pheromones in H. halys. It also provides key candidate genes for bioengineering bioactive aggregation pheromones necessary for the development of technologies for the monitoring and control of H. halys.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

Partial refuges from biological control due to intraspecific variation in protective host traits.

Predicting how much of a host or prey population may be attacked by their natural enemies is fundamental to several subfields of applied ecology, particularly biological control of pest organisms. Hosts or prey can occupy refuges that prevent them from being killed by natural enemies, but habitat or ecological refuges are challenging or impossible to predict in a laboratory setting-which is often where efficacy and specificity testing of candidate biological control agents is done. Here we explore how intraspecific variation in continuous traits of individuals or groups that confer some protection from natural enemy attack-even after the natural enemy has encountered the prey-could provide partial refuges. The size of these trait-based refuges (i.e., the proportion of prey that survive natural enemy encounters due to protective traits) should depend on the relationship between trait values and host/prey susceptibility to natural enemy attack and on how common different trait values are within a host/prey population. These can be readily estimated in laboratory testing of natural enemy impact on target or nontarget prey or hosts as long as sufficient host material is available. We provide a general framework for how intraspecific variation in protective host traits could be integrated into biological control research, specifically with reference to nontarget testing as part of classical biological control programs. As a case study, we exposed different host clutch sizes of target (pest) and nontarget (native species) stink bug (Hemiptera: Pentatomidae) species to a well-studied exotic biocontrol agent, the egg parasitoid Trissolcus japonicus (Hymenoptera: Scelionidae). We predicted that the smallest and largest clutches would occupy trait-based refuges from parasitism. Although we observed several behavioral and reproductive responses to variation in host egg mass size by T. japonicus, they did not translate to increases in host survival large enough to change the conclusions of nontarget testing. We encourage researchers to investigate intraspecific variation in a wider variety of protective host and prey traits and their consequences for refuge size.

Biological Parameters, Phenology and Temperature Requirements of Halyomorpha halys (Hemiptera: Pentatomidae) in the Sub-Mediterranean Climate of Western Slovenia.

In the last decade, the invasive brown marmorated stink bug Halyomorpha halys, native to East Asia, has become one of the most serious pests for agricultural crops worldwide. First detected in Europe (in Switzerland), the insect is now widely found across the European continent and many Eurasian countries. Since its first appearance in Slovenia in 2017 it has caused considerable damage to fruit and vegetable production. Understanding the biology and behavior in the local environmental conditions is of key importance for an effective pest management. Knowledge of the voltinism of the species is crucial to anticipate critical phases of pest development and for adapting control measures that target the vulnerable life stages of the pest. A 3-year study (2019-2021) of H. halys biological parameters was performed outdoors in Nova Gorica (western Slovenia), confirming that in the sub-Mediterranean climate this pest has two overlapping generations per year. The net reproductive rates observed in the studied period indicate growing populations. The highest population growth was recorded in 2019, when the net reproductive rate of increase (R0) reached 14.84 for the summer generation and 5.64 for the overwintering generation. These findings match the current situation in Slovenia, where increasing populations of H. halys and severe crop damage have been observed since 2019.

To Every Thing There Is a Season: Phenology and Photoperiodic Control of Seasonal Development in the Invasive Caucasian Population of the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Heteroptera: Pentatomidae).

Studies on the phenology of local populations of invasive insects are necessary for monitoring and predicting their dispersion. We investigated the phenology of the brown marmorated stink bug, Halyomorpha halys, in the Sochi region (Krasnodar Territory, Russia) from 2018 to 2021 by regular field sampling and dissecting. The results of the sampling suggest that H. halys is at least partially bivoltine in the studied region: the main period of mass oviposition (by the overwintered females) occurs from June to July; the second, much shorter period of egg-laying (by females of the new, i.e., the first generation) occurs in August. Reproductively active individuals (i.e., females with developed ovaries and filled spermatheca and males with filled ectodermal sac) were recorded from the end of May to the beginning of September. Such a seasonal pattern correlated with day length: when the natural photoperiod decreased below the experimentally determined critical day length (15.0−15.5 h), the proportions of females with fully developed ovaries sharply dropped to zero. Both the rate of H. halys pre-adult development and the timing of the induction of winter adult diapause observed under natural conditions fully agreed with the earlier predictions that had been based on the results of laboratory experiments.

Flying over Eurasia: Geographic Variation of Photoperiodic Control of Nymphal Development and Adult Diapause Induction in Native and Invasive Populations of the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Heteroptera: Pentatomidae).

Facultative winter adult diapause in Halyomorpha halys is regulated by a long-day photoperiodic response. Day length also influences nymphal development, which slows down at the critical (near-threshold) day lengths. We compared the photoperiodic responses of one native (Andong, South Korea) and three invasive (Torino, Italy; Basel, Switzerland; and Sochi, Russia) populations in a laboratory common-garden experiment. Nymphs developed and emerging adults were reared at 24 °C in a range of photoperiods with day lengths of 14.0, 14.5, 15.0, 15.5, and 16.0 h. The critical day lengths of the photoperiodic responses of both sexes fell between 14.5 and 15.0 h in the native Korean population and between 15.0 and 15.5 h in three invasive European populations. The differences between the three invasive populations were not significant, despite their distant origins. Moreover, the difference between the Korean and European populations was much smaller than was expected. The microevolution was possibly 'too slow to keep up' with the rapid spread of the invader across Eurasia. It is expected that soon the critical day length of the invasive H. halys populations will gradually change to adapt better to local conditions. At present, the critical day length for diapause induction of 15 h 15 min can be used to model the phenology, further spread, and response to climate change for all European populations of the pest.

Reactive Oxygen Species Initiate Defence Responses of Potato Photosystem II to Sap-Sucking Insect Feeding.

Potato, Solanum tuberosum L., one of the most commonly cultivated horticultural crops throughout the world, is susceptible to a variety of herbivory insects. In the present study, we evaluated the consequence of feeding by the sap-sucking insect Halyomorpha halys on potato leaf photosynthetic efficiency. By using chlorophyll fluorescence imaging methodology, we examined photosystem II (PSII) photochemistry in terms of feeding and at the whole leaf area. The role of reactive oxygen species (ROS) in potato's defence response mechanism immediately after feeding was also assessed. Even 3 min after feeding, increased ROS generation was observed to diffuse through the leaf central vein, probably to act as a long-distance signalling molecule. The proportion of absorbed energy being used in photochemistry (ΦPSII) at the whole leaf level, after 20 min of feeding, was reduced by 8% compared to before feeding due to the decreased number of open PSII reaction centres (qp). After 90 min of feeding, ΦPSII decreased by 46% at the whole leaf level. Meanwhile, at the feeding zones, which were located mainly in the proximity of the leaf midrib, ΦPSII was lower than 85%, with a concurrent increase in singlet-excited oxygen (1O2) generation, which is considered to be harmful. However, the photoprotective mechanism (ΦNPQ), which was highly induced 90 min after feeding, was efficient to compensate for the decrease in the quantum yield of PSII photochemistry (ΦPSII). Therefore, the quantum yield of non-regulated energy loss in PSII (ΦNO), which represents 1O2 generation, remained unaffected at the whole leaf level. We suggest that the potato PSII response to sap-sucking insect feeding underlies the ROS-dependent signalling that occurs immediately and initiates a photoprotective PSII defence response to reduce herbivory damage. A controlled ROS burst can be considered the primary plant defence response mechanism to herbivores.

Attraction of Egg Parasitoids Trissolcus mitsukurii and Trissolcus japonicus to the chemical cues of Halyomorpha halys and Nezara viridula.

Trissolcus mitsukurii and Trissolcus japonicus are two Asian egg parasitoids associated with different pentatomids such as Halyomorpha halys. Adventive populations of T. mitsukurii were found in Northern Italy, suggesting its employment as a biological control agent (BCA) against H. halys. Nevertheless, to reduce the latter's population, T. japonicus was released in Italy. Releasing an exotic parasitoid requires investigating the interaction between the BCA and the environment to avoid negative impacts on the entomofauna of the new habitat. Trissolcus mitsukurii is mainly associated with Nezara viridula in its native area. Therefore, we investigated and compared the ability of female T. mitsukurii and T. japonicus to distinguish between naturally released cues of H. halys and N. viridula. A single parasitoid was exposed to contact kairomones of both pests to evaluate its modifications in orthokinetic and locomotory behaviour. The behaviour of female T. mitsukurii was also tested on synthetic compounds simulating the cues of the two pentatomids. When naturally released cues were used, T. japonicus preferred the traces of H. halys, while T. mitsukurii preferred N. viridula's cues. Moreover, the attraction of T. mitsukurii to N. viridula's cues was confirmed with synthetic cues. Additional studies are needed to judge if this parasitoid can be used as a BCA.

Expressional and functional comparisons of five clustered odorant binding proteins in the brown marmorated stink bug Halyomorpha halys.

Odorant-binding proteins (OBPs) play essential roles in the functioning of insect peripheral olfactory systems. To fully understand the olfactory roles of OBPs in Halyomorpha halys, an important invasive pest found worldwide, we studied the expression and functional characterization of five OBP-associated genes from H. halys that are clustered in the genome. The tissue distribution of the OBP gene cluster suggests that these genes were enriched in nymph and adult antennae, indicating their possible involvement in the chemosensory process. The different expression levels of the five OBPs in nymph and adult antennae suggest that this gene cluster is regulated independently. Ligand-binding experiments have shown similar specificities of these five OBPs towards several organic compounds, including the alarm pheromone of H. halys (trans-2-decenal), the aggregation pheromone of Plautia stali (methyl (2E, 4E, 6Z)-decatrienoate), and plant volatile compounds (e.g., cis-3-hexenyl benzoate and ß-ionone). In particular, trans-2-dodecenal, an alarm pheromone analog, exhibited high affinity to the five OBP proteins and alarm pheromone activity towards H. halys. Thus, this OBP cluster may mediate the response of stink bugs to the both the alarm pheromone and host-related volatiles and could be an interesting target to design novel olfactory regulators for the management of H. halys infestations.

First Report of Native Parasitoids of Halyomorpha halys (Hemiptera: Pentatomidae) in Greece.

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) is an endemic species of East Asia; it was introduced into Europe in 2007. It has a wide range of hosts as it feeds on over 170 host plant species and significantly impacts crop production. In Greece, H. halys causes significant losses in the production of kiwi, peaches, and green beans; thus, control of this species (including biological control) is essential. Here, we focus on the potential impact of native natural enemies of H. halys in Greece. From June to October 2020, we sampled naturally field-laid H. halys egg masses to recover native parasitoids. A total of 20 egg masses of H. halys were collected from infested fields from different locations in northern Greece. Out of 529 eggs, 45 parasitoids managed to hatch successfully. The overall parasitism rate was 8.5%. We found two species of Hymenopteran egg parasitoids attacking H. halys eggs-Anastatus bifasciatus (Geoffrey) (Hymenoptera: Eupelmidae) and Ooencyrtus telenomicida (Vassiliev) (Hymenoptera: Encyrtidae), with the former comprising 58% of all parasitoids that were recovered. These results contribute to the knowledge about the natural enemy community that attacks H. halys in Greece, and the use of these native egg parasitoids in biological control programs may be a viable H. halys management strategy.