Nezara viridula hosts survey around the year cycle. Secondary hosts (SH) from late September to late January; soybean (primary host) from February to late May and under the bark of Eucalyptus trees (diapause) from June to late September. 

Nezara viridula hosts survey around the year cycle. Secondary hosts (SH) from late September to late January; soybean (primary host) from February to late May and under the bark of Eucalyptus trees (diapause) from June to late September. 

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The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized...

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... On average 6.1 zOTUs were found on the eggs of natural N. viridula populations, while on average only 1.2 zOTU was associated with the internal samples. Similarly, low microbial diversity has been found in the midgut of field-collected N. viridula adults (Medina et al., 2018), suggesting that overall microbial diversity associated with N. viridula is low: no culturable bacteria were found in the V1-V3 midgut sections in more than 54% of N. viridula adults collected in the field, while the rest of the stinkbugs were colonized by only a few culturable bacteria like Bacillus, Enterococcus, Micrococcus, Pantoea, Staphylococcus, and Yokenella (Medina et al., 2018). ...
... On average 6.1 zOTUs were found on the eggs of natural N. viridula populations, while on average only 1.2 zOTU was associated with the internal samples. Similarly, low microbial diversity has been found in the midgut of field-collected N. viridula adults (Medina et al., 2018), suggesting that overall microbial diversity associated with N. viridula is low: no culturable bacteria were found in the V1-V3 midgut sections in more than 54% of N. viridula adults collected in the field, while the rest of the stinkbugs were colonized by only a few culturable bacteria like Bacillus, Enterococcus, Micrococcus, Pantoea, Staphylococcus, and Yokenella (Medina et al., 2018). ...
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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. In this study, we assessed the composition and establishment of the microbiome of insect eggs using the Southern green stinkbug Nezara viridula (Hemiptera: Pentatomidae) as a study object. Our results show that the egg‐associated bacterial microbiome is species‐poor and dominated by a few symbionts, particularly the species‐specific obligate Pantoea‐like symbiont.
... Whereas some insects can avoid plant chemical defenses by different behavioral and biochemical mechanisms, [5][6][7] recently laboratory in vitro experiments have suggested that soybean proteases inhibitors can be deactivated by N. viridula's gut microbiota, such as the enterobacteria Yokenella sp. 8 Some specific gut bacteria enable insects to feed on poor diets or digest recalcitrant compounds and increase insect performance. 9,10 Moreover, shifts on bacterial gut communities have allowed contemporary adaptation of insects to overcome new technologies of pest control. ...
... No bacteria were detected in the V1-V3 midgut sections of 60% of field collected N. viridula adults, and the rest were naturally infected with cultivable non-transient bacteria, such as Yokenella sp. 8 This stink bug species houses an obligate symbiont in the caeca (V4) not directly involved in food digestion, which is present in N. viridula from a wide variety of places, including Hawaii, California, Japan, and Brazil. [39][40][41] However, little is known about the biological function of the bacteria present in stink bugs ventricles where digestion is performed (V1-V3 of the midgut). ...
... 38 In field conditions adults of N. viridula have low diverse gut microbiota, and over 60% of individuals lack cultivable bacteria over 10 3 CFU mg −1 gut. 8 However, stink bugs relay upon a single non-cultivable strict symbiont located in a special gut section, and have low diverse and transient microbiota associated to the digestive midgut. 38,45 Therefore, laboratory overcrowding may compromise the immune response of non-social insects as N. viridula, which promote the infection or transfection of bacteria that will change substantially the gut microbiota. ...
Article
Background: The southern green stinkbug (Nezara viridula) is a mayor pest of soybean. However, the mechanism underlying stinkbug resistance to soybean defenses is yet ignored. Although gut bacteria could play an essential role in tolerating plant defenses, most studies testing questions related to insect-plant-bacteria interactions have been performed in laboratory condition. Here we performed experiments in laboratory and field conditions with N.viridula and its gut bacteria, studying gut lipid peroxidaxion levels and cysteine activity in infected and unifected nymphs, testing the hypothesis that feeding on field-grown soybean decreases bacterial abundance in stinkbugs. Results: Gut bacterial abundance and infection ratio were higher in N.viridula adults reared in laboratory than in those collected from soybean crops, suggesting that stinkbugs in field conditions may modulate gut bacterial colonization. Manipulating gut microbiota by infecting stinkbugs with Yokenella sp. showed that these bacteria abundance decreased in field conditions, and negatively affected stinkbugs performance and were more aggressive in laboratory rearing than in field conditions. Infected nymphs that fed on soybean pods had lower mortality, higher mass and shorter development period than those reared in the laboratory, and suggested that field conditions helped nymphs to recover from Yokenella sp. infection, despite of increased lipid peroxidation and decreased cysteine proteases activity in nymphs' guts. Conclusions: Our results demonstrated that feeding on field-grown soybean reduced bacterial abundance and infection in guts of N.viridula and highlighted the importance to test functional activities or pathogenicity of microbes under realistic field conditions prior to establish conclusions on three trophic interactions. This article is protected by copyright. All rights reserved.
... A recent study also showed that a carnivorous diet was preferential for Enterobacteria, including some entomopathogenic bacteria in the gut of plant bugs, compared to an herbivorous diet, which was detrimental to bugs' survival [67]. Enterococcus in stink bugs and other herbivorous insects was reported to be implicated with diet digestion and the detoxification of plant defensive chemicals [68][69][70]. Enterococcus dominated in the gut of generalist herbivore cotton leafworm (Spodoptera littoralis) and was documented to secrete bacteriocin against invading bacteria, providing a defensive function to the host [71]. More empirical tests are needed to validate whether Enterococcus in the gut of assassin bugs plays a similar defensive role for its hosts. ...
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Insects are generally associated with gut bacterial communities that benefit the hosts with respect to diet digestion, limiting resource supplementation, pathogen defense, and ecological niche expansion. Heteroptera (true bugs) represent one of the largest and most diverse insect lineages and comprise species consuming different diets and inhabiting various ecological niches, even including underwater. However, the bacterial symbiotic associations have been characterized for those basically restricted to herbivorous stink bugs of the infraorder Pentatomomorpha. The gut microbiota associated with the megadiverse heteropteran lineages and the implications of ecological and diet variance remain largely unknown. Here, we conducted a bacterial 16S rRNA amplicon sequencing of the gut microbiota across 30 species of true bugs representative of different ecological niches and diets. It was revealed that Proteobacteria and Firmicute were the predominant bacterial phyla. Environmental habitats and diets synergistically contributed to the diversity of the gut bacterial community of true bugs. True bugs living in aquatic environments harbored multiple bacterial taxa that were not present in their terrestrial counterparts. Carnivorous true bugs possessed distinct gut microbiota compared to phytophagous species. Particularly, assassin bugs of the family Reduviidae possessed a characterized gut microbiota predominantly composed of one Enterococcus with different Proteobacteria, implying a specific association between the gut bacteria and host. Overall, our findings highlight the importance of the comprehensive surveillance of gut microbiota association with true bugs for understanding the molecular mechanisms underpinning insect–bacteria symbiosis.
... Some of the bacterial community isolates showed in-vitro β-glucosidase and raffinase activities, which enable the degradation of galactosyl derivatives and increase the digestibility of soybean plants. Enzymes in midgut isolates might also degrade isoflavonoids and deactivate soybean protease inhibitors, helping aphids tolerate soybean defences and feed on the plant (Medina et al., 2018). ...
Chapter
Soybean is a food crop in high demand in Northeast Asia. Besides protein and oil, soybean is also a rich source of health-beneficial secondary metabolites such as flavonoids, terpenes, and alkaloids. The long history of soybean domestication resulted in a rich collection of soybean germplasms, which could be generally categorized as wild, landrace, and cultivated soybeans. Previous research has shown that soybean seeds from diverse genetic backgrounds exhibited different metabolite profiles. Germplasms originating from different geographical regions, i.e., at different latitudes and longitudes, probably experienced different selective pressures and evolved different secondary metabolite profiles. Domestication has generally led to a reduction in secondary metabolite contents in seeds since many of these compounds are related to the bitter taste or other agronomic traits that may hinder the ease of farming and harvest. These selection forces have possibly rendered the different flavors of soybean germplasms. Due to the popularity of soy food products, the post-domestication selection of soybean based on flavor is a common phenomenon. In Northeast Asian countries, soy foods such as soy milk, tofu, and fermented soy products are popular. Based on the consumer preference for the flavors of these products, soybean germplasms with different metabolite profiles are selected for different commercial uses. However, the breeding of soybeans for maximizing health benefits and for the preferred flavors of food products may create contradictions. Industrial methods to remove undesirable flavors and molecular breeding to produce cultivars with desired metabolite profiles may be the solution.
... 13,16,34,35 However, stink bugs are able to avoid plant defenses by inducing digestive proteases inhibitors-insensitive cysteine proteases or by non-transit gut microbiota that deactivates protease inhibitors. 36,37 Currently, in Argentina an infestation rate of four D. furcatus adults per m 2 of soybean at seed development stage (R5) 38 decreases crop yield by around 10%, showing the growing economic burden of this pest. 12 The Principles of Conservation Agriculture have been broadly adopted by Argentine and Brazilian agricultural producers since the 90s. ...
Article
Over the last decades, Argentine and Brazilian farmers have adopted no‐tillage cultivation systems and multiple cropping, which have decreased the abundance of traditional pests, such as Nezara viridula, and favored the development of some stink bugs of secondary importance, like Dichelops furcatus, D. melacanthus and Euschistus heros. No‐till farming leaves the soil undisturbed ‐to mitigate erosion‐, and sufficient crop residue on the field, which is used by D. furcatus as a shelter under unfavorable conditions, and as a shelter against insecticides. Moreover, implementing multiple cropping system increases crop rotation from soybean to corn, and place overwintering adults of D. furcatus in contact with corn seedlings in Spring. Attacks of this stink bug species produce deformation and abortion of corn seedlings, resulting in up to 50% corn yield reduction. The increasing abundance of D. furcatus intensifies the damage on developing pods and seeds of soybean, becoming a primary pest of soybean, and a new pest of corn. Here we summarize the current knowledge about D. furcatus, its biology, life cycle, and geographical distribution pattern in South America. Additionally, we describe the general causes and consequences of D. furcatus as a new pest of corn that emerges from crop stubble. Then, we provide an overview of the chemical control, natural enemies and possible agronomical practices to improve sustainable crop production methods to control this pest. This article is protected by copyright. All rights reserved.
... In some systems, diapause has been shown to reduce microbial diversity to a core subset, as seen in both stink bugs (Nezara viridula) and marine copepods (Arias-Cordero et al., 2012;Datta et al., 2018;Medina et al., 2018). Few studies to date have investigated the role of the microbiome during diapause and other overwintering strategies of insects (Almada, 2015;Dittmer and Brucker, 2021;Liu et al., 2016). ...
Article
The mosquito microbiome is critical to multiple facets of their biology, including larval development and disease transmission. For mosquitoes that reside in temperate regions, periods of diapause are critical to overwintering survival, but how the microbiome impacts this state is unknown. In this study, we compared the midgut microbial communities of diapausing and non-diapausing Culex pipiens and assessed how a reduced midgut microbiome influences diapause preparation. High community variability was found within and between non-diapausing and diapausing individuals, but no specific diapause-based microbiome was noted. Emergence of adult, diapausing mosquitoes under sterile conditions generated low bacterial load (LBL) lines with nearly a 1000-fold reduction in bacteria levels. This reduction in bacterial content resulted in significantly lower survival of diapausing females after two weeks, indicating acquisition of the microbiome in adult females is critical for survival throughout diapause. LBL diapausing females had high carbohydrate levels, but did not accumulate lipid reserves, suggesting an inability to process ingested sugars necessary for lipid accumulation. Expression patterns of select genes associated with mosquito lipid metabolism during diapause showed no significant differences between LBL and control lines, suggesting transcriptional changes may not underlie impaired lipid accumulation. Overall, a diverse, adult-acquired microbiome is critical for diapause in C. pipiens to process sugar reserves and accumulate lipids that are necessary to survive prolonged overwintering.
... Despite an increased interest in the regulatory and physiological aspects of insect diapause [1,[5][6][7][8][9][10][11][12][13][14], the dynamics of host-microbiome interactions during diapause remain underexplored (but see [15][16][17][18]). This contrasts with a growing body of research on hibernating mammals, demonstrating a seasonal remodelling of the gut microbiome between the hibernating and active phases in various species [19][20][21][22]. ...
... While the dominant members of the microbiome remained largely unaffected, diapause seemed to have an impact on many less abundant taxa. This is similar to observations in stinkbugs, where only a non-transient core microbiome was detected in diapausing individuals [17]. In N. vitripennis, about 20% of all bacterial genera were exclusively observed in nondiapausing larvae, indicating that some bacterial taxa may be lost already during preparation for diapause. ...
Article
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Background The life cycles of many insect species include an obligatory or facultative diapause stage with arrested development and low metabolic activity as an overwintering strategy. Diapause is characterised by profound physiological changes in endocrine activity, cell proliferation and nutrient metabolism. However, little is known regarding host-microbiome interactions during diapause, despite the importance of bacterial symbionts for host nutrition and development. In this work, we investigated (i) the role of the microbiome for host nutrient allocation during diapause and (ii) the impact of larval diapause on microbiome dynamics in the parasitoid wasp Nasonia vitripennis , a model organism for host-microbiome interactions. Results Our results demonstrate that the microbiome is essential for host nutrient allocation during diapause in N. vitripennis , as axenic diapausing larvae had consistently lower glucose and glycerol levels than conventional diapausing larvae, especially when exposed to cold temperature. In turn, microbiome composition was altered in diapausing larvae, potentially due to changes in the surrounding temperature, host nutrient levels and a downregulation of host immune genes. Importantly, prolonged larval diapause had a transstadial effect on the adult microbiome, with unknown consequences for host fitness. Notably, the most dominant microbiome member, Providencia sp., was drastically reduced in adults after more than 4 months of larval diapause, while potential bacterial pathogens increased in abundance. Conclusion This work investigates host-microbiome interactions during a crucial developmental stage, which challenges both the insect host and its microbial associates. The impact of diapause on the microbiome is likely due to several factors, including altered host regulatory mechanisms and changes in the host environment.
... Previous studies have mainly focused on stink bug-soybean interactions. [20][21][22][23][44][45][46][47][48][49][50] The ecological behavior and versatility displayed by D. furcatus in host selection make this species an invasive pest that can colonize new niches. This stink bug species makes use of the plant stubble left by the no-tillage system as shelter, which allows them to feed on corn seedlings in spring. ...
Article
BACKGROUND Recently, in temperate and neotropical regions of South America the generalist stink bug Dichelops furcatus (Hemiptera: Pentatomidae) became a new pest of corn (Zea mays) seedlings. Implementation of no‐tillage cultivation system left organic matter covering the soil, which shelters adults of stink bugs during winter. In spring, corn is sowed under soybean stubble and D. furcatus adults start to feed on seedlings. To determine corn‐derived volatile organic compounds (VOCs) that attract this stink bug species, we evaluated stink bug preferences from two corn hybrids with contrast germplasm backgrounds, a temperate and a tropical hybrid. RESULTS Stink bugs preferred to feed on temperate seedlings rather than on the tropical ones. GC–MS and PCA analysis of VOCs suggested that hybrids emitted contrasting blends. Linalool represented 68% of total VOCs emitted from temperate corn, while in the tropical hybrid this compound represented 48%. Olfactometer experiments demonstrated that linalool was attractive to stink bugs. However, 2 h of D. furcatus attack induced emission of 14 additional VOCs in temperate seedlings, and olfactometer bioassay and blend of VOCs emission suggested that perceived volatiles by stink bugs induced feeding avoidance. The increment of VOCs emission was associated with the induction of JA, JA‐Ile, ABA, and IAA, and decreasing of SA concentrations. CONCLUSION This is the first time showing a complete profile of defensive phytohormones induced by stink bugs feeding on corn, and further demonstrating that a blend of corn seedling‐associated VOCs, mainly composed by linalool, modulates D. furcatus adults' behavior and feeding preferences. This article is protected by copyright. All rights reserved.
... Compared to the relatively large body of information on the stink bug obligate symbionts, few data are available concerning the other members of the gut microbial community and their interactions with the main symbionts. Studies of the gut microbiome have been conducted on adult individuals of few pentatomid species, like H. halys and N. viridula, revealing that the bacterial community of the crypt-harboring midgut portion (V4) is largely dominated by the primary symbionts (Kenyon et al., 2015;Medina et al., 2018), but yet colonized by other bacterial phyla in many stink bug hosts. For instance, in a survey on seven pentatomid species, different Actinobacteria were detected in the terminal midgut portion, mostly in the genera Corynebacterium, Dietzia, Citricoccus, Mycobacterium, Propionibacterium, and Streptomyces. ...
... In the other midgut compartments (named V1-V3), the primary symbionts are much less abundant (Medina et al., 2018), and many other bacteria have been found. For example, in the digestive tract of the red-banded stink bug Piezodorus guildinii (Westwood) several bacteria putatively involved in nutrient provision and digestion have been identified, such as Klebsiella oxytoca, Clostridium butyricum, and Citrobacter farmeri, along with the candidate primary symbiont Pantoea dispersa (Husseneder et al., 2017). ...
... For example, in the digestive tract of the red-banded stink bug Piezodorus guildinii (Westwood) several bacteria putatively involved in nutrient provision and digestion have been identified, such as Klebsiella oxytoca, Clostridium butyricum, and Citrobacter farmeri, along with the candidate primary symbiont Pantoea dispersa (Husseneder et al., 2017). The analysis of different N. viridula populations from Brazil, Hawaii, California and Japan, confirmed that the terminal midgut ventriculus was dominated by a single bacterial type of the family Enterobacteraceae, while in the remaining compartments of the gut other Enterobacteria and Enterococci were detected (e.g., Klebsiella pneumoniae, Enterococcus faecalis, and Yokenella sp.), possibly being involved in detoxification of the food source (Medina et al., 2018). Many stink bugs are recognized as vectors of different plant pathogens, which often colonize the digestive tract, representing an additional component of the gut microbiota (Mitchell, 2004;Esquivel et al., 2010;Esquivel and Medrano, 2014;Medrano et al., 2016). ...
Article
Full-text available
The family Pentatomidae (Hemiptera: Heteroptera) includes several invasive stink bug species capable to attack a large number of wild and cultivated plants, causing several damages to different crops. Pentatomids rely on obligate symbiotic associations with bacteria of the family Enterobacteriaceae , mainly of the genus Pantoea . A distinctive trait of these associations is the transmission route: during oviposition, females smear egg masses with symbiont-containing secretions, which are ingested by newly hatched nymphs, allowing the symbiont to pass through their digestive tract and establish in the crypts of the posterior midgut. Preventing newborns from orally acquiring symbionts seriously affects their fitness and survival. This symbiont inheritance process can be manipulated to develop innovative pest control measures by sterilization of egg masses prior to nymph hatching. This review summarizes the recent knowledge advances concerning the gut primary symbionts of pentatomids, with a specific focus on the most troubling pest species for agriculture. Current understanding of host colonization dynamics in pentatomids is presented, as well as the phenotypic effects determined in different insect species by the alteration of vertical transmission. Details on the current knowledge on the whole bacterial communities accompanying primary symbionts are analyzed. The recent research exploiting the perturbation of symbiont acquisition by pentatomid nymphs is discussed, by considering published work on laboratory and field trials with several active substances. These translational strategies are presently regarded as promising for limiting the populations of many important pentatomid pests in a sustainable way.
... The presence of symbiotic bacteria was reported in the gut tissue of the aphid Acyrthosiphon pisum, and removal of these bacteria reduced certain metabolites, including essential amino acids 38 . Similarly, the bacterial community colonizing the midgut of N. viridula could play a role in nutritional status and deactivation of soybean chemical defenses 39 . It has been proposed that stink bug saliva could carry yeasts and bacteria that eventually grow within the seeds 40 . ...
Article
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The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.