Protective Geometry and Reproductive Anatomy as Candidate Determinants of Clutch Size Variation in Pentatomid Bugs.

AbstractMany animals lay their eggs in clusters. Eggs on the periphery of clusters can be at higher risk of mortality. We asked whether the most commonly occurring clutch sizes in pentatomid bugs could result from geometrical arrangements that maximize the proportion of eggs in the cluster's interior. Although the most common clutch sizes do not correspond with geometric optimality, stink bugs do tend to lay clusters of eggs in shapes that protect increasing proportions of their offspring as clutch sizes increase. We also considered whether ovariole number, an aspect of reproductive anatomy that may be a fixed trait across many pentatomids, could explain observed distributions of clutch sizes. The most common clutch sizes across many species correspond with multiples of ovariole number. However, there are species with the same number of ovarioles that lay clutches of widely varying size, among which multiples of ovariole number are not overrepresented. In pentatomid bugs, reproductive anatomy appears to be more important than egg mass geometry in determining clutch size uniformity. In addition, our analysis demonstrates that groups of animals with little variation in ovariole number may nonetheless lay a broad range of clutch shapes and sizes.

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.

Current distribution and voltinism of the brown marmorated stink bug, Halyomorpha halys, in Switzerland and its response to climate change using a high-resolution CLIMEX model.

Climate change can alter the habitat suitability of invasive species and promote their establishment. The highly polyphagous brown marmorated stinkbug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), is native to East Asia and invasive in Europe and North America, damaging a wide variety of fruit and vegetable crops. In Switzerland, crop damage and increasing populations have been observed since 2017 and related to increasing temperatures. We studied the climatic suitability, population growth, and the number of generations under present and future climate conditions for H. halys in Switzerland, using a modified version of the bioclimatic model package CLIMEX. To address the high topographic variability in Switzerland, model simulations were based on climate data of high spatial resolution (approx. 2 km), which significantly increased their explanatory power, and identified many more climatically suitable areas in comparison to previous models. The validation of the CLIMEX model using observational records collected in a citizen science initiative between 2004 and 2019 revealed that more than 15 years after its accidental introduction, H. halys has colonised nearly all bioclimatic suitable areas in Switzerland and there is limited potential for range expansion into new areas under present climate conditions. Simulations with climate change scenarios suggest an extensive range expansion into higher altitudes, an increase in generations per year, an earlier start of H. halys activity in spring and a prolonged period for nymphs to complete development in autumn. A permanent shift from one to two generations per year and the associated population growth of H. halys may result in increasing crop damages in Switzerland. These results highlight the need for monitoring the spread and population development in the north-western part of Switzerland and higher altitudes of the valleys of the south.

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.

Discrimination between Eggs from Stink Bugs Species in Europe Using MALDI-TOF MS.

In the current paper, we used a method based on stink bug egg-protein immobilization on filter paper by drying, followed by post-(storage and shipping) extraction in acidified acetonitrile containing matrix, to discriminate between nine different species using MALDI-TOF MS. We obtained 87 correct species-identifications in 87 blind tests using this method. With further processing of the unblinded data, the highest average Bruker score for each tested species was that of the cognate reference species, and the observed differences in average Bruker scores were generally large and the errors small except for Capocoris fuscispinus, Dolycoris baccarum, and Graphosoma italicum, where the average scores were lower and the errors higher relative to the remaining comparisons. While we observed clear discrimination between the nine species using this method, Halyomorpha halys and Piezodorus lituratus were more spectrally related than the other pairwise comparisons.

A temporal trophic shift from primary parasitism to facultative hyperparasitism during interspecific competition between two coevolved scelionid egg parasitoids.

Understanding competition between scelionid parasitoids that exploit the same host may provide insight into strategies that allow coexistence on a shared resource. Competition studies typically focus on interactions between native and exotic parasitoids that do not share an evolutionary history; however, coevolved parasitoids may be more likely to demonstrate strategies to avoid or exploit a shared resource. We examined intrinsic and extrinsic competition between Asian Trissolcus japonicus (Ashmead) and T. cultratus (Mayr) (Hymenoptera: Scelionidae) associated with Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) that share an evolutionary history. Interspecific interactions were assessed by providing parasitized egg masses to each species at various intervals post-parasitism, and measuring host acceptance, developmental suitability, and guarding behaviour. Trissolcus japonicus showed high acceptance of parasitized hosts up to 72 h following oviposition by T. cultratus, despite a very poor developmental outcome. In contrast, T. cultratus generally avoided ovipositing in H. halys eggs containing T. japonicus early-instar larvae but did not avoid parasitizing H. halys that contained eggs and third instar larvae. The adaptive value of this behaviour was supported by developmental outcome: T. cultratus outcompeted T. japonicus eggs but not early-instar larvae, and a trophic shift occurred wherein T. cultratus developed as a facultative hyperparasitoid on third instar T. japonicus larvae. Trissolcus japonicus guarded egg masses 8-12× longer and displayed more aggressive interactions than T. cultratus, suggesting T. japonicus is the superior extrinsic competitor. Development as a facultative hyperparasitoid provided a competitive niche for Asian T. cultratus and confirms its instrinsic competitive superiority. This also occurs in a biologically distinct European population of T. cultratus, suggesting that facultative hyperparasitism as a competitive strategy is retained in geographically separated populations that have not coevolved with H. halys or T. japonicus.

Assessing the Distribution of Exotic Egg Parasitoids of Halyomorpha halys in Europe with a Large-Scale Monitoring Program.

The brown marmorated stink bug Halyomorpha halys is an invasive agricultural pest with a worldwide distribution. Classical biological control has been identified as the most promising method to reduce the populations of H. halys. Adventive populations of two candidates for releases, Trissolcus japonicus and Trissolcus mitsukurii, have recently been detected in Europe. To assess their distribution and abundance, a large-scale survey was performed. From May to September 2019, a wide area covering northern Italy and parts of Switzerland was surveyed, highlighting the expanding distribution of both Tr. japonicus and Tr. mitsukurii. Within four years after their first detection in Europe, both species have rapidly spread into all types of habitats where H. halys is present, showing a wide distribution and continuous expansion. Both exotic Trissolcus showed high levels of parasitism rate towards H. halys, while parasitization of non-target species was a rare event. The generalist Anastatus bifasciatus was the predominant native parasitoid of H. halys, while the emergence of native scelionids from H. halys eggs was rarely observed. The presence of the hyperparasitoid Acroclisoides sinicus was also recorded. This study provided fundamental data that supported the development of the first inoculative release program of Tr. japonicus in Europe.

Next-generation biological control: the need for integrating genetics and genomics.

Biological control is widely successful at controlling pests, but effective biocontrol agents are now more difficult to import from countries of origin due to more restrictive international trade laws (the Nagoya Protocol). Coupled with increasing demand, the efficacy of existing and new biocontrol agents needs to be improved with genetic and genomic approaches. Although they have been underutilised in the past, application of genetic and genomic techniques is becoming more feasible from both technological and economic perspectives. We review current methods and provide a framework for using them. First, it is necessary to identify which biocontrol trait to select and in what direction. Next, the genes or markers linked to these traits need be determined, including how to implement this information into a selective breeding program. Choosing a trait can be assisted by modelling to account for the proper agro-ecological context, and by knowing which traits have sufficiently high heritability values. We provide guidelines for designing genomic strategies in biocontrol programs, which depend on the organism, budget, and desired objective. Genomic approaches start with genome sequencing and assembly. We provide a guide for deciding the most successful sequencing strategy for biocontrol agents. Gene discovery involves quantitative trait loci analyses, transcriptomic and proteomic studies, and gene editing. Improving biocontrol practices includes marker-assisted selection, genomic selection and microbiome manipulation of biocontrol agents, and monitoring for genetic variation during rearing and post-release. We conclude by identifying the most promising applications of genetic and genomic methods to improve biological control efficacy.

Can Anastatus bifasciatus Be Used for Augmentative Biological Control of the Brown Marmorated Stink Bug in Fruit Orchards?

The generalist egg parasitoid Anastatus bifasciatus (Geoffroy) (Hymenoptera: Eupelmidae) is the most prevalent egg parasitoid of the invasive Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) in Europe. To assess its efficacy against the pest H. halys and to validate the potential risks for non-target species in a realistic field setting, inundative releases were conducted over three consecutive years in four fruit orchards in Switzerland and Italy. In total, more than 4300 A. bifasciatus females were released, which was equivalent to 11,000 to 26,000 females per hectare, depending on distances between trees in each orchard. Parasitism of freeze-killed sentinel H. halys eggs achieved with the current release strategy was on average 6% (range: 2%-16%) and considered not high enough to effectively suppress the pest. However, the overall impact of A. bifasciatus on the mortality of H. halys eggs was likely underestimated. If pre-imaginal parasitoid mortality (3.3%) and host feeding (6%) are added to the observed parasitism (6%), the actual induced mortality of H. halys eggs may reach more than 15%. Parasitism of lepidopteran non-target species reached an average of 8% and thus, some degree of non-target parasitism after mass releases may be expected. To quantify the impact of the parasitoids in the orchards more precisely, naturally laid egg masses should be used in future trials to include host-finding cues of the host and host plants, and larger scale releases with potentially higher densities of parasitoids should be considered.

Using the egg parasitoid Anastatus bifasciatus against the invasive brown marmorated stink bug in Europe: can non-target effects be ruled out?

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), has been causing massive damage to various fruit and vegetable crops after its arrival in the USA, and more recently in Europe. To provide an alternative control measure to pesticides, the native egg parasitoid Anastatus bifasciatus (Geoffroy) (Hymenoptera: Eupelmidae) was considered as a candidate biological control agent for inundative releases in Europe. In the risk assessment study presented here, all nine heteropteran and 14 out of 19 tested lepidopteran non-target species produced viable A. bifasciatus offspring. The proportion of A. bifasciatus females producing offspring did not differ between non-target and target for 19 out of the 28 non-target species. Larger host eggs corresponded to increased female-biased sex ratio of the offspring as well as an increase in size, particularly for females, with hind tibia lengths varying from 645.5 ± 46 to 1084 ± 28.5 µm. Larger females were also found to have higher offspring production and increased life expectancy. The results of this study confirmed the polyphagous nature of A. bifasciatus and suggest that a number of non-target species, including Lepidoptera of conservation interest, may be attacked in the field. Thus, non-target effects cannot entirely be ruled out, but more information is needed from semi-field and field studies to fully assess potential environmental risks due to inundative releases of this native parasitoid.

Host specificity of Asian parasitoids for potential classical biological control of Drosophila suzukii.

The Asian spotted wing drosophila, Drosophila suzukii, has recently become a serious pest of soft fruits in Europe. Classical biological control through the introduction of larval parasitoids from its native range in Asia is presently being considered. However, host specificity of potential biological control agents has to be determined to avoid releasing species that may have unintended non-target impacts. Larvae of six different European non-target fly species and the target D. suzukii were exposed either on diet or blueberries to three Asian larval parasitoids, Asobara japonica, Leptopilina japonica, and Ganaspis cf. brasiliensis, and one European species, Leptopilina heterotoma. Asobara japonica showed the lowest specificity, attacking and developing in all Drosophilidae. Leptopilina japonica successfully parasitized two non-target Drosophilidae, D. melanogaster and D. subobscura, with one singly progeny emerging from D. immigrans. Ganaspis cf. brasiliensis had the highest level of specificity but variations occurred between two geographical populations tested. A Japanese population was strictly specific to D. suzukii, whereas another population from China parasitized D. suzukii, D. melanogaster and sporadically D. subobscura. The European L. heterotoma successfully developed in D. melanogaster, D. subobscura and occasionally in D. immigrans, but nearly all eggs and larvae in D. suzukii were encapsulated. These results show that Ganaspis cf. brasiliensis is the species with the highest potential for biological control, but more studies are needed on its taxonomic status and the existence of biotypes or cryptic species varying in their specificity before field releases can be conducted in Europe.

The parasitoid complex of D. suzukii and other fruit feeding Drosophila species in Asia.

Drosophila suzukii is an invasive fly of East Asian origin that has become a serious fruit pest worldwide. Classical biological control through the introduction of parasitoids from Asia could help reduce populations of D. suzukii in invaded regions. Little is known about the native parasitoids of the fly in Asia. Therefore, surveys for larval parasitoids of D. suzukii were carried out in China and Japan between 2015 and 2017. Parasitoids of D. suzukii and other fruit-inhabiting drosophilids (D. pulchrella and D. subpulchrella) that are probably attacked by the same parasitoid complex were found in four Chinese provinces and four Japanese prefectures. Larval parasitoids were obtained at most sites where D. suzukii was found, with parasitism varying from 0.0 to 75.6%. At least eight parasitoid species were reared. The most abundant and frequent parasitoids were the Figitidae Ganaspis cf. brasiliensis and Leptopilina japonica, but another Leptopilina species and at least five Braconidae species belonging to the genera Areotetes, Asobara and Tanycarpa were obtained in low numbers. Due to its likely restricted host range, the most promising parasitoid for biological control is Ganaspis cf. brasiliensis. However, its exact specificity and taxonomic status require future research.

Seasonal parasitism and host specificity of Trissolcus japonicus in northern China.

The brown marmorated stink bug, Halyomorpha halys (Stål), native to China, Japan, and Korea, has emerged as a harmful invasive pest of a variety of crops in North America and Europe. The Asian egg parasitoid Trissolcus japonicus has been identified as the most promising agent for classical biological control of invasive H. halys populations. A 4-year study evaluated the fundamental and ecological host ranges of T. japonicus as well as its phenology and impact on H. halys populations in fruit orchards in its native range in northern China. In laboratory no-choice tests, developmental suitability of eight non-target host species for T. japonicus was demonstrated by the successful production of progeny on the majority (>85%) of non-target host species tested. In field-collected, naturally laid egg masses, T. japonicus was the most abundant parasitoid associated with H. halys and Dolycoris baccarum, but was also sporadically found in Plautia crossota. Furthermore, it was regularly reared from sentinel egg masses of Menida violacea, Arma chinensis, and Carbula eoa. The only species that did not support development in the laboratory and field was Cappaea tibialis. Besides the benefit of having a high impact on H. halys populations in Northern China, the risk assessment conducted in the area of origin indicates that native Pentatomidae in North America and Europe could be negatively impacted by T. japonicus. Whether the benefits of T. japonicus outweigh the possible risks will have to be evaluated based on the outcome of additional host range studies in the two invaded regions.

An exotic parasitoid provides an invasional lifeline for native parasitoids.

The introduction of an exotic species may alter food webs within the ecosystem and significantly affect the biodiversity of indigenous species at different trophic levels. It has been postulated that recent introduction of the brown marmorated stinkbug (Halyomorpha halys (Stål)) represents an evolutionary trap for native parasitoids, as they accept H. halys egg masses as a host but produce no viable progeny. Interspecific interactions between European egg parasitoid, Trissolcus cultratus (Mayr), and an Asian parasitoid, Trissolcus japonicus (Ashmead), were assessed by providing egg masses to T. cultratus at various time intervals following the initial parasitization by T. japonicus. The suitability of the host for the parasitoid development was re-assessed by providing T. cultratus with fresh and frozen egg masses of various ages. The likelihood of T. cultratus being able to attack previously parasitized egg masses was determined by assessing the duration of egg mass guarding behavior by T. japonicus following parasitization. The results of experiments examining the interspecific interactions between a native European egg parasitoid, T. cultratus, and an Asian parasitoid, T. japonicus (a candidate for the biological control of H. halys), showed that the native species can act as facultative hyperparasitoid of the exotic one. Although this is only possible during certain stages of T. japonicus development, the presence of the introduced parasitoid may reduce the impact of the evolutionary trap for indigenous parasitoid species. There is a possibility that the occurrence of facultative hyperparasitism between scelionid parasitoids associated with stinkbugs is common. This resulting intraguild predation could promote conservation and stabilization of natural communities by impacting the diversity and population dynamics of native stinkbugs and their parasitoids (e.g., by allowing native parasitoids to avoid wasting reproductive effort on unsuitable hosts), or reduce success of biological control programs (e.g., by reducing the population size of the exotic parasitoids).

Attraction of Halyomorpha halys (Hemiptera: Pentatomidae) haplotypes in North America and Europe to baited traps.

Halyomorpha halys is a global invasive species, native to Southeast Asia, that is threatening agriculture in invaded regions. Our objectives were to: 1) establish the attractiveness of semiochemical stimuli paired with field-deployed traps in Europe (Greece, Hungary, Italy, and Switzerland), compared with Maryland, USA, and 2) identify H. halys haplotypes recovered from traps at each location. We found qualitatively identical patterns of capture between sites located across Europe and in Maryland, USA. In both regions, captures of H. halys adults indicated a synergistic response to traps baited with the two component H. halys aggregation pheromone, and pheromone synergist, methyl (2E, 4E, 6Z)-decatrienoate when compared with either individually. Haplotype diversity in Europe based on trapped specimens was much greater than the USA, with five new haplotypes described here, probably indicating ongoing invasion and re-introduction of H. halys. By contrast, a single, previously identified haplotype was trapped in Maryland, USA, representing a single introduction. All H. halys haplotypes responded to each semiochemical in apparent proportion to their frequency in the overall population based on independently derived information from prior work. Taken together, these data suggest that pheromone-based technology will be of global utility for the monitoring of this important invasive species.

Non-crop plants used as hosts by Drosophila suzukii in Europe.

The invasive spotted wing drosophila Drosophila suzukii, a fruit fly of Asian origin, is a major pest of a wide variety of berry and stone fruits in Europe. One of the characteristics of this fly is its wide host range. A better knowledge of its host range outside cultivated areas is essential to develop sustainable integrated pest management strategies. Field surveys were carried out during two years in Italy, the Netherlands and Switzerland. Fruits of 165 potential host plant species were collected, including mostly wild and ornamental plants. Over 24,000 D. suzukii adults emerged from 84 plant species belonging to 19 families, 38 of which being non-native. Forty-two plants were reported for the first time as hosts of D. suzukii. The highest infestations were found in fruits of the genera Cornus, Prunus, Rubus, Sambucus and Vaccinium as well as in Ficus carica, Frangula alnus, Phytolacca americana and Taxus baccata. Based on these data, management methods are suggested. Ornamental and hedge plants in the vicinity of fruit crops and orchards can be selected according to their susceptibility to D. suzukii. However, the widespread availability and abundance of non-crop hosts and the lack of efficient native parasitoids suggest the need for an area-wide control approach.