Differential effects of plant-beneficial fungi on the attraction of the egg parasitoid Trissolcus basalis in response to Nezara viridula egg deposition.

There is increasing evidence that plant-associated microorganisms play important roles in defending plants against insect herbivores through both direct and indirect mechanisms. While previous research has shown that these microbes can modify the behaviour and performance of insect herbivores and their natural enemies, little is known about their effect on egg parasitoids which utilize oviposition-induced plant volatiles to locate their hosts. In this study, we investigated how root inoculation of sweet pepper (Capsicum annuum) with the plant-beneficial fungi Beauveria bassiana ARSEF 3097 or Trichoderma harzianum T22 influences the olfactory behaviour of the egg parasitoid Trissolcus basalis following egg deposition by its host Nezara viridula. Olfactometer assays showed that inoculation by T. harzianum significantly enhanced the attraction of the egg parasitoid, while B. bassiana had the opposite effect. However, no variation was observed in the chemical composition of plant volatiles. Additionally, fitness-related traits of the parasitoids (wasp body size) were not altered by any of the two fungi, suggesting that fungal inoculation did not indirectly affect host quality. Altogether, our results indicate that plant inoculation with T. harzianum T22 can be used to enhance attraction of egg parasitoids, which could be a promising strategy in manipulating early plant responses against pest species and improving sustainable crop protection. From a more fundamental point of view, our findings highlight the importance of taking into account the role of microorganisms when studying the intricate interactions between plants, herbivores and their associated egg parasitoids.

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.

Microbe-mediated alterations in floral nectar: consequences for insect parasitoids.

Floral nectar is frequently colonized by microbes among which bacteria and yeasts are the most abundant. These microbes have the ability to alter nectar characteristics with consequences for the whole community of flower-visiting insects. Recent research carried out on natural enemies of insect herbivores has shown that microbe-mediated changes in nectar traits can influence the foraging behavior and life history traits of parasitoids. The production of microbial volatile organic compounds can affect the attraction of parasitoids to nectar, while changes in sugar and amino acid composition can impact their longevity. Future research should focus on understanding the effects of nectar microbial colonization on parasitoid reproduction, with a specific emphasis on the interactions among different microbial taxa known to co-occur in floral nectar. Overall, this review highlights the importance of considering the role of nectar-inhabiting microbes in shaping the interactions between parasitoids and their food resources.

Finding an egg in a haystack: variation in chemical cue use by egg parasitoids of herbivorous insects.

Egg parasitoids of herbivorous insects use an interplay of short- and long-range chemical cues emitted by hosts and host plants to find eggs to parasitize. Volatile compounds that attract egg parasitoids can be identified via behavioral assays and used to manipulate parasitoid behavior in the field for biological control of herbivorous pests. However, how and when a particular cue will be used varies over the life of an individual, as well as at and below species level. Future research should expand taxonomic coverage to explore variation in chemical cue use in more natural, dynamic settings. More nuanced understanding of the variability of egg parasitoid host-finding strategies will aid in disentangling the underlying genetics and further enhancing biological control.

Competitive interactions in insect parasitoids: effects of microbial symbionts across tritrophic levels.

Competition for hosts is a common ecological interaction in insect parasitoids. In the recent years, it has become increasingly evident that microorganisms can act as 'hidden players' in parasitoid ecology. In this review, we propose that parasitoid competition should take into consideration the microbial influence. In particular, we take a tritrophic perspective and discuss how parasitoid competition can be modulated by microorganisms associated with the parasitoids, their herbivore hosts, or the plants attacked by the herbivores. Although research is still in its infancy, recent studies have shown that microbial symbionts can modulate the contest outcome. The emerging pattern is that microorganisms not only affect the competitive traits of parasitoids but also the fighting arena (i.e. the herbivore host and its food plant), in which competition takes place. We have also identified important gaps in the literature that should be addressed in future studies to advance our understanding about parasitoid competition.

Nectar-Inhabiting Bacteria Affect Olfactory Responses of an Insect Parasitoid by Altering Nectar Odors.

Floral nectar is ubiquitously colonized by a variety of microorganisms among which yeasts and bacteria are the most common. Microorganisms inhabiting floral nectar can alter several nectar traits, including nectar odor by producing microbial volatile organic compounds (mVOCs). Evidence showing that mVOCs can affect the foraging behavior of insect pollinators is increasing in the literature, whereas the role of mVOCs in altering the foraging behavior of third-trophic level organisms such as insect parasitoids is largely overlooked. Parasitoids are frequent visitors of flowers and are well known to feed on nectar. In this study, we isolated bacteria inhabiting floral nectar of buckwheat, Fagopyrum esculentum (Polygonales: Polygonaceae), to test the hypothesis that nectar bacteria affect the foraging behavior of the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae) via changes in odors of nectar. In behavioral assays, we found that T. basalis wasps are attracted toward nectar fermented by 4 out of the 14 bacterial strains isolated, which belong to Staphylococcus epidermidis, Terrabacillus saccharophilus (both Firmicutes), Pantoea sp. (Proteobacteria), and Curtobacterium sp. (Actinobacteria). Results of chemical investigations revealed significant differences in the volatile blend composition of nectars fermented by the bacterial isolates. Our results indicate that nectar-inhabiting bacteria play an important role in the interactions between flowering plants and foraging parasitoids. These results are also relevant from an applied perspective as flowering resources, such as buckwheat, are largely used in agriculture to promote conservation biological control of insect pests.

Chemical Ecology of Floral Resources in Conservation Biological Control.

Conservation biological control aims to enhance populations of natural enemies of insect pests in crop habitats, typically by intentional provision of flowering plants as food resources. Ideally, these flowering plants should be inherently attractive to natural enemies to ensure that they are frequently visited. We review the chemical ecology of floral resources in a conservation biological control context, with a focus on insect parasitoids. We highlight the role of floral volatiles as semiochemicals that attract parasitoids to the food resources. The discovery that nectar-inhabiting microbes can be hidden players in mediating parasitoid responses to flowering plants has highlighted the complexity of the interactions between plants and parasitoids. Furthermore, because food webs in agroecosystems do not generally stop at the third trophic level, we also consider responses of hyperparasitoids to floral resources. We thus provide an overview of floral compounds as semiochemicals from a multitrophic perspective, and we focus on the remaining questions that need to be addressed to move the field forward.

Diversity and composition of the microbiome associated with eggs of the Southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae).

Although microbial communities of insects from larval to adult stage have been increasingly investigated in recent years, little is still known about the diversity and composition of egg-associated microbiomes. In this study, we used high-throughput amplicon sequencing and quantitative PCR to get a better understanding of the microbiome of insect eggs and how they are established using the Southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) as a study object. First, to determine the bacterial community composition, egg masses from two natural populations in Belgium and Italy were examined. Subsequently, microbial community establishment was assessed by studying stinkbug eggs of different ages obtained from laboratory strains (unlaid eggs collected from the ovaries, eggs less than 24 h old, and eggs collected 4 days after oviposition). Both the external and internal egg-associated microbiomes were analyzed by investigating egg washes and surface-sterilized washed eggs, respectively. Eggs from the ovaries were completely devoid of bacteria, indicating that egg-associated bacteria were deposited on the eggs during or after oviposition. The bacterial diversity of deposited eggs was very low, with on average 6.1 zero-radius operational taxonomic units (zOTUs) in the external microbiome and 1.2 zOTUs in internal samples of egg masses collected from the field. Bacterial community composition and density did not change significantly over time, suggesting limited bacterial growth. A Pantoea-like symbiont previously found in the midgut of N. viridula was found in every sample and generally occurred at high relative and absolute densities, especially in the internal egg samples. Additionally, some eggs harbored a Sodalis symbiont, which has previously been found in the abdomen of several insects, but so far not in N. viridula populations. We conclude that the egg-associated bacterial microbiome of N. viridula is species-poor and dominated by a few symbionts, particularly the species-specific obligate Pantoea-like symbiont.

Contrasting reproductive traits of competing parasitoids facilitate coexistence on a shared host pest in a biological control perspective.

BACKGROUND: Interspecific competition in insect parasitoids is an important ecological phenomenon that has relevant implications for biological pest control. To date, interspecific intrinsic (=larval) competition has been intensively studied, while investigations on extrinsic (=adult) competition have often lagged behind. In this study we examined the role played by parasitoid reproductive traits and host clutch size on the outcome of extrinsic competition between Trissolcus basalis (Wollaston) and Ooencyrtus telenomicida (Vassiliev), two egg parasitoids of the pest Nezara viridula (L). Laboratory experiments were conducted by allowing both parasitoid species to exploit an egg mass made of 10, 20, 30, or 40 hosts through single or simultaneous releases. Furthermore, under field conditions, egg masses consisting of 10 or 40 hosts were exposed in a tomato crop in order to validate laboratory investigation. RESULTS: The results show that the egg mass size is an important predictor of extrinsic competition in our study system as a higher proportion of T. basalis emerged from large egg masses, while O. telenomicida dominated in small egg masses. Analysis of reproductive traits of parasitoid species indicates that T. basalis has superior abilities in host exploitation compared with O. telenomicida. CONCLUSIONS: We found that contrasting reproductive traits of two competing egg parasitoid species facilitate coexistence on a shared stink bug host. This work also highlights the importance to consider extrinsic competitive interactions between parasitoid species in a biological control perspective. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Ecology of Hyperparasitoids.

Hyperparasitoids are some of the most diverse members of insect food webs. True hyperparasitoids parasitize the larvae of other parasitoids, reaching these larvae with their ovipositor through the herbivore that hosts the parasitoid larva. During pupation, primary parasitoids also may be attacked by pseudohyperparasitoids that lay their eggs on the parasitoid (pre)pupae. By attacking primary parasitoids, hyperparasitoids may affect herbivore population dynamics, and they have been identified as a major challenge in biological control. Over the past decades, research, especially on aphid- and caterpillar-associated hyperparasitoids, has revealed that hyperparasitoids challenge rules on nutrient use efficiency in trophic chains, account for herbivore outbreaks, or stabilize competitive interactions in lower trophic levels, and they may use cues derived from complex interaction networks to locate their hosts. This review focuses on the fascinating ecology of hyperparasitoids related to how they exploit and locate their often inconspicuous hosts and the insect community processes in which hyperparasitoids are prominent players.

Impact of parasitoid-associated polydnaviruses on plant-mediated herbivore interactions.

Insect herbivores interact via plant-mediated interactions in which one herbivore species induces changes in plant quality that affects the performance of a second phytophagous insect that shares the food plant. These interactions are often asymmetric due to specificity in induced plant responses to herbivore attack, amount of plant damage, elicitors in herbivore saliva and plant organ damaged by herbivores. Parasitoids and their symbiotic polydnaviruses alter herbivore physiology and behaviour and may influence how plants respond to parasitized herbivores. We argue that these phenomena affect plant-mediated interactions between herbivores. We identify that the extended phenotype of parasitoid polydnaviruses is an important knowledge gap in interaction networks of insect communities.

Plant-phenotypic changes induced by parasitoid ichnoviruses enhance the performance of both unparasitized and parasitized caterpillars.

There is increasing awareness that interactions between plants and insects can be mediated by microbial symbionts. Nonetheless, evidence showing that symbionts associated with organisms beyond the second trophic level affect plant-insect interactions are restricted to a few cases belonging to parasitoid-associated bracoviruses. Insect parasitoids harbour a wide array of symbionts which, like bracoviruses, can be injected into their herbivorous hosts to manipulate their physiology and behaviour. Yet, the function of these symbionts in plant-based trophic webs remains largely overlooked. Here, we provide the first evidence of a parasitoid-associated symbiont belonging to the group of ichnoviruses which affects the strength of plant-insect interactions. A comparative proteomic analysis shows that, upon parasitoid injection of calyx fluid containing ichnovirus particles, the composition of salivary glands of caterpillars changes both qualitatively (presence of two viral-encoded proteins) and quantitatively (abundance of several caterpillar-resident enzymes, including elicitors such as glucose oxidase). In turn, plant phenotypic changes triggered by the altered composition of caterpillar oral secretions affect the performance of herbivores. Ichnovirus manipulation of plant responses to herbivory leads to benefits for their parasitoid partners in terms of reduced developmental time within the parasitized caterpillar. Interestingly, plant-mediated ichnovirus-induced effects also enhance the performances of unparasitized herbivores which in natural conditions may feed alongside parasitized ones. We discuss these findings in the context of ecological costs imposed to the plant by the viral symbiont of the parasitoid. Our results provide intriguing novel findings about the role played by carnivore-associated symbionts on plant-insect-parasitoid systems and underline the importance of placing mutualistic associations in an ecological perspective.

Escaping the evolutionary trap: Can size-related contest advantage compensate for juvenile mortality disadvantage when parasitoids develop in unnatural invasive hosts?

The quality of hosts for a parasitoid wasp may be influenced by attributes such as host size or species, with high quality for successful development usually coincident with high quality for larger offspring. This is not always the case: for the Scelionid wasp Trissolcus basalis, oviposition in eggs of the Brown Marmorated Stink Bug, Halyomorpha halys, rather than of the normal host, the Southern Green Stink Bug, Nezara viridula, leads to lower offspring survival, but survivors can be unusually large. Adult female T. basalis engage in contests for host access. As larger contestants are typically favoured in contests between parasitoids, the larger size of surviving offspring may compensate for the mortality of others. We construct a general game-theoretic model to explore whether size advantage can sustain a maternal preference to utilize a more deadly host species. We find that size advantage alone is unlikely to sustain a shift in host preference, yet such an outcome is possible when size asymmetries act simultaneously with advantages in host possession (ownership effect). Halyomorpha halys is an invasive pest of major agro-economic importance in Europe and the Americas, and use of its eggs as hosts by native parasitoids such as T. basalis has been seen as an evolutionary trap due to their high developmental mortality. Our model suggests that the recently discovered effect of host choice on offspring size may provide an escape from the trap via effects on contest biology of T. basalis which could foster a more stable association with H. halys. An evolutionary shift in the reproductive value of H. halys could increase the efficiency of T. basalis as a biological control agent of this invasive stink bug pest.

Influence of parasitoid-associated viral symbionts on plant-insect interactions and biological control.

Insect parasitoids have evolved symbiotic interactions with several viruses and thousands of parasitoid species have established mutualistic associations with polydnaviruses (PDVs). While PDVs have often been described as virulence factors allowing development of immature parasitoids inside their herbivore hosts, there is increasing awareness that PDVs can affect plant-insect interactions. We review recent literature showing that PDVs alter not only host physiology, but also feeding patterns and composition of herbivore's oral secretions. In turn PDV-induced changes in herbivore phenotype affect plant responses to herbivory with consequences ranging from differential expression of plant defense-related genes to wider ecological effects across multiple trophic levels. In this opinion paper we also highlight important missing gaps to fully understand the role of PDVs and other parasitoid-associated viral symbionts in a plant-insect interaction perspective. Because PDVs negatively impact performance and survival of herbivore pests, we conclude arguing that PDV genomes offer potential opportunities for biological control.

Trichoderma harzianum Strain T22 Modulates Direct Defense of Tomato Plants in Response to Nezara viridula Feeding Activity.

Plant growth-promoting fungi belonging to genus Trichoderma are known to help plants when dealing with biotic stressors by enhancing plant defenses. While beneficial effects of Trichoderma spp. against plant pathogens have long been documented, fewer studies have investigated their effect on insect pests. Here, we studied the impact of Trichoderma root colonization on the plant defense responses against stink bug feeding attack. For this purpose, a model system consisting of tomato plant, Solanum lycopersicum cv Dwarf San Marzano, Trichoderma harzianum strain T22 and the southern green stink bug, Nezara viridula, was used. We firstly determined stink bug performance in terms of relative growth rate and survival on tomato plants inoculated by T. harzianum T22. Then, we evaluated relative expression of plant defense-related genes on inoculated plants induced by stink bug feeding. We found evidence that T. harzianum T22 affects tomato defense responses against N. viridula nymphs leading to reduction of growth rate. Our results also showed that T. harzianum T22 enhances plant direct defenses by an early increase of transcript levels of jasmonic acid marker genes. Yet this effect was time-dependent and only detected 8 h after herbivore induction. Taken together, our findings provide better understanding on the mechanisms underlying tomato induced resistance against herbivorous stink bugs.

Microbial Symbionts of Parasitoids.

Parasitoids depend on other insects for the development of their offspring. Their eggs are laid in or on a host insect that is consumed during juvenile development. Parasitoids harbor a diversity of microbial symbionts including viruses, bacteria, and fungi. In contrast to symbionts of herbivorous and hematophagous insects, parasitoid symbionts do not provide nutrients. Instead, they are involved in parasitoid reproduction, suppression of host immune responses, and manipulation of the behavior of herbivorous hosts. Moreover, recent research has shown that parasitoid symbionts such as polydnaviruses may also influence plant-mediated interactions among members of plant-associated communities at different trophic levels, such as herbivores, parasitoids, and hyperparasitoids. This implies that these symbionts have a much more extended phenotype than previously thought. This review focuses on the effects of parasitoid symbionts on direct and indirect species interactions and the consequences for community ecology.

Exploiting chemical ecology to manage hyperparasitoids in biological control of arthropod pests.

Insect hyperparasitoids are fourth trophic level organisms that commonly occur in terrestrial food webs, yet they are relatively understudied. These top-carnivores can disrupt biological pest control by suppressing the populations of their parasitoid hosts, leading to pest outbreaks, especially in confined environments such as greenhouses where augmentative biological control is used. There is no effective eco-friendly strategy that can be used to control hyperparasitoids. Recent advances in the chemical ecology of hyperparasitoid foraging behavior have opened opportunities for manipulating these top-carnivores in such a way that biological pest control becomes more efficient. We propose various infochemical-based strategies to manage hyperparasitoids. We suggest that a push-pull strategy could be a promising approach to 'push' hyperparasitoids away from their parasitoid hosts and 'pull' them into traps. Additionally, we discuss how infochemicals can be used to develop innovative tools improving biological pest control (i) to restrict accessibility of resources (e.g. sugars and alternative hosts) to primary parasitoid only or (ii) to monitor hyperparasitoid presence in the crop for early detection. We also identify important missing information in order to control hyperparasitoids and outline what research is needed to reach this goal. Testing the efficacy of synthetic infochemicals in confined environments is a crucial step towards the implementation of chemical ecology-based approaches targeting hyperparasitoids. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Understanding insect foraging in complex habitats by comparing trophic levels: insights from specialist host-parasitoid-hyperparasitoid systems.

Insects typically forage in complex habitats in which their resources are surrounded by non-resources. For herbivores, pollinators, parasitoids, and higher level predators research has focused on how specific trophic levels filter and integrate information from cues in their habitat to locate resources. However, these insights frequently build specific theory per trophic level and seldom across trophic levels. Here, we synthesize advances in understanding of insect foraging behavior in complex habitats by comparing trophic levels in specialist host-parasitoid-hyperparasitoid systems. We argue that resources may become less apparent to foraging insects when they are member of higher trophic levels and hypothesize that higher trophic level organisms require a larger number of steps in their foraging decisions. We identify important knowledge gaps of information integration strategies by insects that belong to higher trophic levels.

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.