Southern Green Stinkbug Nezara Viridula Research Articles

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.

Reduction in Insect Attachment Caused by Different Nanomaterials Used as Particle Films (Kaolin, Zeolite, Calcium Carbonate)

In the present investigation, we compared the reduction in attachment ability of the southern green stinkbug Nezara viridula (Hemiptera: Pentatomidae) to glass induced by three different nanoparticle (kaolin, zeolite, and calcium carbonate) films. Using traction force experiments, behavioral experiments, and scanning electron microscopy observations, we analyzed the insect attachment ability and linear speed on untreated and treated glass with the three particle films. The three nanomaterials strongly reduced insect attachment ability mainly owing to contamination of attachment pads. The ability to reduce insect attachment was different for the three tested particle films: kaolin and zeolite induced a significantly higher reduction in N. viridula safety factor than calcium carbonate. The coating of the surface was more uniform and compact in kaolin and zeolite compared to calcium carbonate particle film. Moreover, kaolin and zeolite particles can more readily adhere to N. viridula attachment devices, whereas calcium carbonate particles appeared less adherent to the cuticular surface compared to the two aluminosilicate (kaolin and zeolite) particles. Only the application of kaolin reduced insect linear speed during locomotion. Nanoparticle films have a great potential to reduce insect attachment ability and represent a good alternative to the use of insecticides for the control of pentatomid bugs and other pest insects.

Silencing of Double-Stranded Ribonuclease Improves Oral RNAi Efficacy in Southern Green Stinkbug Nezaraviridula.

Variability in RNA-interference (RNAi) efficacy among different insect orders poses a big hurdle in the development of RNAi-based pest control strategies. The activity of double-stranded ribonucleases (dsRNases) in the digestive canal of insects can be one of the critical factors affecting oral RNAi efficacy. Here, the involvement of these dsRNases in the southern green stinkbug Nezaraviridula was investigated. First, the full sequence of the only dsRNase (NvdsRNase) in the transcriptome of N. viridula was obtained, followed by an oral feeding bioassay to evaluate the effect of NvdsRNase-silencing on oral RNAi efficacy. The NvdsRNase was first silenced in nymphs by NvdsRNase-dsRNA injections, followed by exposure to an artificial diet containing a lethal αCop-specific dsRNA. A significantly higher mortality was observed in the NvdsRNase-silenced nymphs when placed on the dsαCop-containing diet (65%) than in the dsGFP injected and dsαCop fed control (46.67%). Additionally, an ex vivo dsRNA degradation assay showed a higher stability of dsRNA in the saliva and midgut juice of NvdsRNase-silenced adults. These results provide evidence for the involvement of NvdsRNase in the reduction of oral RNAi efficacy in N. viridula. This information will be useful in further improving potential RNAi-based strategies to control this pest.

Front Cover Image, Volume 77, Issue 1

The front cover image is based on the Research Article RNAi‐mediated mortality in southern green stinkbug Nezara viridula by oral delivery of dsRNA by Rohit Sharma et al., https://doi.org/10.1002/ps.6017.image

RNAi-mediated mortality in southern green stinkbug Nezara viridula by oral delivery of dsRNA.

The southern green stinkbug, Nezara viridula (Hemiptera: Pentatomidae), is an important emerging polyphagous pest infesting soybean in the United States, Brazil and Argentina. The indiscriminate use of synthetic insecticides to control stinkbugs has limited the effectiveness of current management strategies. Alternatively, RNA interference (RNAi) has emerged as a novel mode of action to control pests in an eco-friendly manner. Here, we assessed the potential of RNAi technology by oral delivery of double-stranded RNA (dsRNA) for the control of N. viridula. Initially, ten candidate genes were tested by microinjection assay to select the best target genes for oral delivery. Seven genes resulted in more than 90% mortality after microinjection. To evaluate RNAi efficacy by oral delivery of dsRNA, five genes were tested by feeding the insects on gene-specific dsRNA mixed with an artificial diet. Significant mortality of 43% and 45% was observed after 14 days of treatment with dsαCop and dsvATPase A, respectively. To elucidate the lower RNAi efficacy via oral delivery of dsRNA, ex vivo dsRNA degradation in the saliva and the midgut juice was performed, which indicated that the reduced RNAi efficacy is accompanied by a rapid degradation of dsRNA by digestive secretions. This study proves that RNAi can be triggered by orally delivered dsRNA in N. viridula and can be exploited to control this economically important pest. The reduced stability of dsRNA in saliva and midgut that was observed indicates a need to further improve RNAi efficacy, for example by use of specific formulations.

Entrapment of Bradysia paupera (Diptera: Sciaridae) by Phaseolus vulgaris (Fabaceae) plant leaf

Flies of the genus Bradysia (Diptera, Sciaridae) are considered as major insect pests of greenhouse-grown horticultural crops. The ability of hooked trichomes of the French bean Phaseolus vulgaris to impale and entrap herbivorous insects thus resulting in insect reduced longevity, reproduction and larval mortality is well known. The present study investigates under laboratory conditions the ability of hooked trichomes of bean leaves to entrap fungus gnats Bradysia paupera, in order to estimate the reduction of their population. We characterized the entrapment mechanism of hooked trichomes towards B. paupera using cryo-scanning electron microscopy, and evaluated the silicon distribution in hooked trichomes with the energy dispersive X-ray microanalysis. We evaluated the trapping efficiency of hooked trichomes in fertilized and unfertilized bean plants towards B. paupera, in comparison with insects feeding on the plant leaf such as black bean aphid Aphis fabae and young stages of the southern green stinkbug Nezara viridula. For B. paupera, we recorded about 30% of entrapped insects in unfertilized plants. Considering the number of entrapped insects in relation to the leaf surface, the percentage of entrapped insect was higher in unfertilized than in fertilized plants having lower density of hooked trichomes. The presence of P. vulgaris plants in greenhouses could represent a useful method in integrated pest management to reduce Bradysia spp. population.

Development of efficient RNAi in Nezara viridula for use in insecticide target discovery.

Stink bugs are an emerging pest in many regions of the world but their molecular biology is still poorly understood. While several transcriptomes are available, the lack of validated gene manipulation tools like RNA interference (RNAi) in species such as the southern green stinkbug Nezara viridula precludes the characterization of individual genes in vivo. Such tools are particularly useful in performing high-throughput screens to search for essential genes that can be prioritized as potential insecticide targets. Here, we developed and optimized an efficient RNAi in N. viridula for use in insecticide target discovery and beyond. The visible marker Sex combs reduced and the essential gene Actin were used to verify the usability and efficiency of RNAi by microinjection at both the adult and nymphal stages, respectively, with nymphal approach presenting significant advantages. Following validation, RNAi was then used to measure lethality following the knockdown (KD) of two genes that are known insecticide targets, Chitin synthase, and Acetyl-CoA carboxylase. The KD of each gene resulted in >75% corrected mortality. These results indicate that RNAi is an effective tool in N. viridula and set a benchmark to evaluate potential targets in future RNAi screens aimed at insecticide target discovery.

Female competition for availability of males in insects: the Nezara viridula (Linnaeus, 1758) model.

Multimodal communication in solitary stinkbugs enables them to meet, mate and copulate. Many plant-dwelling species exchange information during the calling phase of mating behavior using substrate-borne vibratory signals. A female-biased gender ratio induces rivalry and competition for a sexual partner. Female competition for males, first described among Heteroptera in three stinkbug species, revealed species specific differences and opened the question of plasticity in individually emitted temporal and frequency signal characteristics during calling and rival alternation. To address this question and gain an insight into the mechanisms underlying stinkbug female rivalry, we compared the characteristics of alternated signals in the southern green stinkbug Nezara viridula (Linnaeus, 1758) (Hemiptera: Pentatomidae). Compared to male rivalry, female rivalry is more complex, lasts longer and runs through successive phases by a combination of different song types. The male pheromone triggers alternation between females, producing song pulses that occasionally overlap each other. One female initiates the rivalry by changing individual pulses into pulse trains of three different types. The competing female alternates with pulses of changed temporal characteristics at lower levels of rivalry and by varying the frequency characteristics of pulse trains at higher levels. During female rivalry, the male either stops responding or occasionally emits calling and courtship signals in response to the female that has produced signals of steady temporal characteristics. Female rivalry shows complex and species specific patterns of information exchange at different levels with a broad-range variation of temporal and frequency characteristics of, until now, unidentified vibratory emissions.

Structure and biomechanics of the antennal grooming mechanism in the southern green stink bug Nezara viridula

Insects devote a large amount of time to self-groom to remove foreign material, especially from their sensory appendages. Using various microscopy techniques and behavioural experiments on intact and ablated insects, the present study investigates the antennal grooming of the southern green stinkbug Nezara viridula, which represents a serious pest of different crops in most areas of the world. The antennal grooming behaviour encompasses an action of scraping involving the tibial comb complex (tibial comb + fossula) of both forelegs, generally followed by the tibial comb complex grooming of one leg using the tarsal hairy adhesive pad of the opposite leg (rubbing). From our observations, we can exclude a role in the antennal grooming of other structures such as the foretibial apparatus, while we show an involvement of this last structure in repositioning the stylets inside the labium. The external and internal morphology (cryo-scanning and transmission electron microscopy) and the evidence for the presence of large proportions of the elastic protein resilin (confocal laser scanning microscopy) in some parts of both the tibial comb complex and the foretibial apparatus are shown, and their functional roles are discussed. For the first time we demonstrated here the multipurpose role of the basitarsal hairy adhesive pad that is involved in both antennal grooming and adhesion to the substrate.

Prey range of the predatory ladybird Cryptolaemus montrouzieri

The prey range of Cryptolaemus montrouzieri was studied in the laboratory to investigate whether the mealybug destroyer can contribute to the suppression of other pest insects besides mealybugs and to assess its potential impact on non-mealybug populations as part of an environmental risk assessment for its use in biological control. Prey tested in these experiments were: tobacco aphid Myzus persicae nicotianae (Sulzer)(Hemiptera: Aphididae), pea aphid Acyrthosiphon pisum (Harris)(Hemiptera: Aphididae), tobacco whitefly Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae), southern green stinkbug Nezara viridula (L.)(Hemiptera: Pentatomidae) eggs, western flower thrips Frankliniella occidentalis (Pergande)(Thysanoptera: Thripidae), two-spotted ladybird Adalia bipunctata (L.)(Coleoptera: Coccinellidae) eggs and eggs of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae). Larval survival was high to moderate when C. montrouzieri was provided with hemipteran prey and poor to zero when the ladybirds were provided with non-hemipteran prey. Females reared on M. persicae and A. pisum produced similar numbers of eggs as their counterparts fed the citrus mealybug Planococcus citri (Risso)(Hemiptera: Pseudococcidae), but fecundity was significantly lower when the ladybirds were reared on B. tabaci nymphs or on A. bipunctata eggs. Prey species that were found to be less suitable for immature development of C. montrouzieri could still be an adequate food source for reproduction and survival of adult ladybirds. For example, only 8 % of the predator larvae reached the adult stage when provided with A. bipunctata eggs, but females that had developed on eggs of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) and that were supplied with A. bipunctata eggs from adult emergence on, were only 35 % less fecund than females provided with mealybugs in their adult life. The results are discussed in relation to the development of a suitable methodology for prey/host range testing in the framework of an environmental risk assessment for arthropod natural enemies.

Search behaviour of two hemipteran species using vibrational communication

Abstract The ability of conspecifics to recognize and locate each other in the environment depends on the efficiency of intraspecific communication. We compared the mate searching strategies of southern green stinkbug Nezara viridula (male searches for a continuously calling female) and the leafhopper Aphrodes makarovi (partners form a precisely coordinated duet). Males of both species were tested on plants in playback experiments. One leaf was vibrated with unaltered conspecific female signals or with various conspecific signals using modified temporal parameters. The results showed that the onset of searching was faster in A. makarovi than in N. viridula. Changes in temporal parameters of female replies had negative effect on the searching behaviour of A. makarovi. Males located the source of longer female replies faster than the short female call and they failed to locate the source of a female reply with temporal parameters outside the species-specific values. In contrast, in N. viridula, searching males successfully located also the source of a female song with parameters outside the species-specific values. The results are discussed with regard to male behavioural strategies in species with different vibrational communication systems and different male mating investment.

Obligate association with gut bacterial symbiont in Japanese populations of the southern green stinkbug Nezara viridula (Heteroptera: Pentatomidae)

The southern green stinkbug Nezara viridula (Linnaeus) has a number of sac-like outgrowths, called crypts, in a posterior section of the midgut, wherein a specific bacterial symbiont is harbored. In previous studies on N. viridula from Hawaiian populations, experimental elimination of the symbiont caused few fitness defects in the host insect. Here we report that N. viridula from Japanese populations consistently harbors the same gammaproteobacterial gut symbiont, but, in contrast with previous work, experimental sterilization of the symbiont resulted in severe nymphal mortality, indicating an obligate host–symbiont relationship. Considering worldwide host–symbiont association and these experimental data, we suggest that N. viridula is generally and obligatorily associated with the gut symbiont, but that the effect of the symbiont on host biology may be different among geographic populations. Possible environmental factors that may affect the host–symbiont relationship are discussed.

Identification of the first neuropeptides from the CNS of Hemiptera: CAPA peptides of the southern green stinkbug Nezara viridula (L.)

A direct mass spectrometric investigation of nerve homologs of the abdominal perisympathetic organs was employed to reveal the first and complete sequences of CAPA peptides from a hemipteran species, the southern green stinkbug Nezara viridula. Side-chain fragmentations allowed the assignment of internal Leu/Ile; on-plate acetylation was used to distinguish between the mass-related Lys and Gln. The following sequences were obtained: DQLFPFPRV-NH 2 (CAPA-PVK-1), EQLIPFPRV-NH 2 (CAPA-PVK-2), and NGSAGNGGLWFGPRL/I-NH 2 (CAPA-PK). CAPA-PVKs are associated with the regulation of diuresis in insects, and identification of those native to a hemipteran will provide the experimental basis to better understand regulation of water balance in this family of insects.

EFFECTS OF AN ENCAPSULATED FORMULATION OF LAMBDA-CYHALOTHRIN ON NEZARA VIRIDULA AND ITS PREDATOR PODISUS MACULIVENTRIS (HETEROPTERA: PENTATOMIDAE)

Insecticidal effects of an encapsulated formulation of lambda-cyhalothrin on the southern green stinkbug Nezara viridula (L.) and one of its predators, Podisus maculiventris (Say), were investigated in the laboratory. Both pentatomids were exposed to the insecticide via contaminated drinking water and by residual contact. Nymphs and adults of N. viridula were more susceptible to the insecticide than nymphs of P. maculiventris, both by ingestion and contact exposure. For the respective ways of exposure, LC50 values calculated for P. maculiventrisfourth instars were 30-190 times and 3-13 times higher than those of N. viridulafourth instars. Insecticidal activity of the pyrethroid by ingestion was 6-10 times greater against nymphs of N. viridula than against adults of the pest. In both the ingestion and residual contact experiments, nymphs of P. maculiventris recovered from initial knockdown. LC50 values for predator nymphs increased 1.7- to 2.7-fold between 24 and 48 h after the start of the experiment. Recovery from knockdown was not observed in N. viridula. The data from the current laboratory study suggest that encapsulated lambda-cyhalothrin may be effective for controlling the southern green stinkbug with little adverse effects on the predator P. maculiventris, but field experiments are needed to confirm this. Possible reasons for the differential toxicity of the insecticide to both pentatomids are discussed.

PREDATION BY PODISUS MACULIVENTRIS ON DIFFERENT LIFE STAGES OF NEZARA VIRIDULA

Abstract Predation capacity of fourth instars and male and female adults of Podisus maculiventris (Say) (Heteroptera: Pentatomidae) on the different life stages of the southern green stinkbug Nezara viridula (L.) (Heteroptera: Pentatomidae) was measured in the laboratory. Both nymphal and adult predators displayed high predation rates on eggs, nymphs and adults of the southern green stinkbug. However, developmental times of fourth instar P. maculiventris on eggs or nymphal instars of N. viridula were longer than on fifth instars of the cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), suggesting that N. viridula is suboptimal prey for the spined soldier bug. Preference experiments in which fourth instar P. maculiventris were given a choice between fourth instars of N. viridula and fifth instars of S. littoralis indicated that the stinkbugs were less vulnerable to predation than the caterpillars, mainly because of their greater agility. The potential role of P. maculiventris in a...

Temporal and spectral properties of the songs of the southern green stink bug Nezara viridula (L.) from Slovenia.

Substrate born songs of the southern green stinkbug Nezara viridula (L.) from Slovenia were recorded and analysed. The male calling song is composed of narrow-band regularly repeated single pulses and of broad-band frequency modulated pulses grouped into pulse trains. The female calling song is characterised by broad-band pulsed and narrow-band non-pulsed pulse trains. A frequency modulated pre-pulse precedes the narrow-band pulse train. A frequency-modulated post-pulse usually follows the pulse train of the male courtship song. The male calling song triggers broad-band pulse trains of the female courtship song. The female also produces a repelling low-frequency vibration that inhibits male calling and courtship. The male rival song is characterised by prolonged pulses with a typical frequency modulation.