Phylogeographic relationships of the southern green stink bug Nezara viridula (Hemiptera: Pentatomidae)

Abstract The sequences of 16S rDNA, cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) genes from nine field collections (seven provinces in China: Guangxi, Hubei, Guangdong, Guizhou, Zhejiang, Hunan, Jiangxi, and also southern and northern part of Iran), plus the sequences of Africa, Europe, Americas and Japan obtained form GenBank were used to re-analyse the genetic variation in the southern green stink bug Nezara viridula (Linnaeus). The phylogeographic re-analysis by using four algorithms (NJ, MP, ML and Bayesian) showed three main lineages. The Iranian haplotypes fell into lineage II formed from Europe and America, rather than in lineage III from Asia; the Chinese haplotypes fell into the Asian clade. Our results suggested that African and non-African gene pools have been isolated since the Miocene era with the molecular clock calibrations for Heteroptera mtDNA, and not since the Pliocene as mentioned previously. The corresponding age of the separation of the eastern and western Asia clades is estimated to be 4.0–1.6 million years ago, coinciding with the Pliocene–Pleistocene epoch and with acute rising events in the Tibet Plateau locates in the western China, which may have formed the barriers observed today.     

Temporal and numerical patterns of reproductive behavior in the southern green stink bug,Nezara viridula (Hemiptera: Pentatomidae)

Through hourly observations of 370 individuals in two 11-day test periods, the temporal and numerical patterns of mating and oviposition behavior inNezara viridula (L.) were investigated in the laboratory. There was large variation in copulatory durations (1–165 hr), number of copulations per individual (1–9), and timein copulo prior to oviposition (1–176 hr). Number of copulations and mean copulatory duration were negatively correlated for both sexes. Number of copulations was positively correlated with percent of total test time spent mating. Neither males nor females exhibited definitive refractory periods. Oviposition followed a marked diel periodicity.

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Zusammenfassung Das Paarungs- und Eiablageverhalten vonNezara viridula (Hemiptera: Pentatomidae) wurde im Labor beobachtet. Es wurden 370 markierte. Wanzen während zwei 11-tägigen Versuchen berücksichtigt. Die in kleinen Käfigen eingeschlossenen Insekten wurden einmal pro Stunde kontrolliert. Die jeweilige Kopulationsdauer für alle Paare wurde registriert, wie auch die Trennungszeiten der Individuen. Die Dauer der Kopulation schwankte von 1 bis 165 Stunden; die Individuen paarten sich 1 bis 9 mal; bei den Weibchen betrug die Gesamtpaarungszeit vor der Eiablage 1 bis 176 Stunden. Für die beiden Geschlechter verringerte sich jede einzelne Kopulationsdauer, sobald die Paarungshäufigkeit zunahm. Je niedriger die Kopulationsrate war, desto geringer war auch die gesamte Kopulationszeit (Fig. 2). Es wurden weder für die Männchen noch für die Weibchen bestimmte Inaktivitätszeiten registriert; manche blieben für längere Zeit ohne weitere Paarungen, während andere sich sofort wieder paarten. Die Eiablage fand hauptsächlich von 18.00–21.00 Uhr statt (Fig. 3).

     

Digestive proteolytic activity in the pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m₁ and m₃, respectively, and maximum chymotrypsin and trypsin activities were observed in m₃. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.     

Monitoring stink bugs (Hemiptera: Pentatomidae) in mid-Atlantic apple and peach orchards

Pyramid traps coated with industrial safety yellow exterior latex gloss enamel paint and baited with Euschistus spp. aggregation pheromone, methyl (2E,4Z)-decadienoate captured more stink bugs than all other baited and unbaited trap types in both apple and peach orchards in 2002 and 2003. Commercial sources of dispensers of methyl (2E,4Z)-decadienoate deployed in association with pyramid traps had a significant impact on trap captures. Captures in pyramid traps were four-fold greater when baited with lures from IPM Technologies, Inc. (Portland, OR) than with lures from Suterra (Bend, OR). Variation in yellow pyramid trap color (industrial safety yellow and standard coroplast yellow) and material (plywood, plastic, and masonite) did not affect trap captures. Brown stink bug was the predominant species captured (58%), followed by dusky stink bug, Euschistus tristigmus (Say) (20%); green stink bug, Acrosternum hilare (Say) (14%); and other stink bugs (Brochymena spp. and unidentified nymphs) (8%). Captures in baited pyramid traps were significantly correlated with tree beating samples in both managed and unmanaged apple orchards and with sweep netting samples in the unmanaged apple orchard. However, problems associated with trapping mechanisms of pyramid trap jar tops and jar traps likely resulted in reduced captures in baited traps. Improved trapping mechanisms must be established to develop an effective monitoring tool for stink bugs in mid-Atlantic orchards.     

Studies on the adult polymorphism in the southern green stink bug,Nezara viridula (hemiptera: Pentatomidae)

Polymorphism in adult colour pattern of Nezara viridula is determined on the genetic basis. The basic colour patterns of adult are classified into four types, i. e. G, O, R and F. No appreciable differences between these types were observed in respect to various physiological traits of nymphs and adults, except that type G seems to be superior in reproductive ability but to be inferior in ability of surviving winters at least to types R and F. Inter-generation changes in percentage frequency of G type were examined from 1959 to 1967 covering more than 30 generations. Percentage frequencies of G types prior to the severe winter of 1962–3 fluctuated greatly around a mean of 87.9%, while they did to a lesser extent than before with a mean of 85.0% after the winter. Unexpected high percentages of G type were recorded frequently in summer generations, viz. 1st and 2nd. On the other hand, the relative frequencies of G and O types decreased after hibernation in contrast to the increases in those of F and R. This sort of changes in genetic composition related to the winter of 1962–3 was observed in several populations segregated from each other. This seasonal alternation of selective activity in the environments is considered to be responsible for retention of the polymorphism. Alternative possible causes, i. e. difference in habitat preference, non-random mating and selective predation by predators among polymorphs, may safely be rejected as irrelevant to the mechanism in maintaining polymorphism. The polymorphism of this insect seems to be in a transient state rather than balanced one contributing little to population regulation, but the persistence of N. viridula in the periphery range may be assisted by retention of the polymorphism.     

Toxicity of Chromobacterium subtsugae to southern green stink bug (Heteroptera: Pentatomidae) and corn rootworm (Coleoptera: Chrysomelidae)

Diabrotica spp. (Coleoptera: Chrysomelidae) beetles and southern green stink bugs, Nezara viridula (L.) (Heteroptera: Pentatomidae), are pests on corn, Zea mays L., and soybean, Glycine max (L.) Merr., as well as on cucurbits. Control of these insects has depended on chemicals. An alternative to chemical control is the use of biologicals. Use of bacteria, fungi, viruses, pheromones, and metabolites to control these insects can potentially improve resistance management and reduce pesticide use. Other than Bacillus thuringiensis Berliner, few bacteria have been discovered that are lethal to either of these pests. Chromobacterium subtsugae Martin et al., a newly described bacterium that is known to be toxic to Colorado potato beetle, Leptinotarsa decemlineata (Say), larvae, was found to be toxic to both diabroticite adult beetles and southern green stink bug adults. In laboratory assays, toxins produced by these bacteria kill 80-100% of the adults of two species of diabroticite beetles, Diabrotica undecimpunctata howardi Barber and Diabrotica virgifera virgifera LeConte, and 100% of southern green stink bug adults within 6 d. For green stink bug, live bacteria were not needed for toxicity.     

Delayed maturity associated with southern green stink bug (Heteroptera: Pentatomidae) injury at various soybean phenological stages

Delayed maturity in soybean, Glycine max (L.) Merr., occurred in response to infestation by southern green stink bug, Nezara viridula (L.), in 4 yr of field studies. Maturity delays followed stink bug infestation that occurred only during the pod set and filling stages (R3-R5.5), and infestations at R3-4 and R5 resulted in delayed maturity more consistently than did infestation at R5.5. Infestation levels of six stink bugs per 0.3 m of row for 7-14 d generally were required to delay soybean maturity. The greatest impact on seed yield and quality parameters followed stink bug infestations that occurred during R3-R5.5, which corresponded closely with the periods of infestation that resulted in delayed maturity. If both delayed maturity and yield reduction are considered, the pod elongation through late pod filling stages were most critical for protecting soybeans from southern green stink bugs.     

Possible egg positioning and gluing behavior by ovipositing southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae)

From March 2002 to January 2004, a colony of southern green stink bug, Nezara viridula (L.), was kept in the laboratory, and its oviposition behavior was observed. During oviposition, soon after the egg is expelled, the female touches the egg mass with the dorsal surface of the last tarsomere; this movement of one leg of the hind pair occurred once. This small component of the oviposition behavior of this pentatomid is little known and not yet fully understood; it may help to position and glue the newly deposited egg to the others.     

Cotton boll age influences feeding preference by brown stink bug (Heteroptera: Pentatomidae)

Cotton plants were infested with brown stink bug, Euschistus servus (Say), to define cotton boll age classes (based on heat unit accumulation beyond anthesis) that are most frequently injured during each of the initial 5 wk of flowering. Bolls from each week were grouped into discrete age classes and evaluated for the presence of stink bug injury. Brown stink bug injured significantly more bolls of age class B (approximately 165-336 heat units), age class C (approximately 330-504 heat units), and age class D (approximately 495-672 heat units) during the initial 3 wk in both years and in week 5 in 2002 compared with other boll ages. Generally, the frequency of injured bolls was lowest in age class A (< or = 168 heat units) during these periods. The preference by brown stink bug for boll age classes B, C, and D within a week was similar when ages were combined across all 5 wk. Based on these data, bolls that have accumulated 165.2 through 672 heat units beyond anthesis (approximately 7-27-d-old) are more frequently injured by brown stink bug when a range of boll ages are available. The boll ages in our studies corresponded to a boll diameter of 1.161-3.586 cm with a mid-range of 2.375 cm. A general protocol for initiating treatments against stink bugs is to sample bolls for evidence of injury as an indicator of presence of infestations in cotton. Sampling bolls within a defined range, which is most likely to be injured, should improve the precision of this method in detecting economic stink bug infestations in cotton.     

The effect of vibratory disturbance on sexual behaviour of the southern green stink bug Nezara viridula (Heteroptera, Pentatomidae)

The effect of vibratory disturbance on sexual behaviour and substrate-borne sound communication of the southern green stink bug, Nezara viridula L. was studied. Disturbance signals do not change the time N. viridula males need to locate the source of vibratory signals, but decrease the number of males responding with the calling and courtship song to calling females. Female N. viridula proceed calling during stimulation with disturbance signals but some of them change the song rhythm by skipping one or more signal intervals or emitting the repelling signals. The number of females which change the dominant frequency of the calling song decreases proportionally with increasing differences between the dominant frequency of the disturbance signals and the emitted female calling song. Variation of the song dominant frequency probably serves females to avoid interference by increasing the signal to noise ratio. Signal duration and repetition rate do not change significantly when the female is stimulated with the disturbance signals. This indicates that frequency shift by calling females is the main strategy for reducing interference by competitive signalers in N. viridula vibrational communication.     

Treatment thresholds for stink bugs (Hemiptera: Pentatomidae) in cotton

The green stink bug, Acrosternum hilare (Say), the southern green stink bug, Nezara viridula (L), and the brown stink bug, Euschistus servus (Say), were predominant phytophagous Pentatomidae detected during 1995-1997 in cotton in South Carolina. These species occurred in similar numbers in conventional and transgenic cotton 'NuCOTN33B', containing the gene for expression of CryIA(c) delta-endotoxin of Bacillus thuringiensis Berliner variety kurstaki. Adult stink bugs moved into cotton from wild and cultivated alternate hosts during July, and reproducing populations usually were detected in cotton from late July into September. Applications of either methyl parathion (0.56 kg [AI]/ha) directed for stink bugs or lambda-cyhalothrin (0.037 kg [AI]/ha) or cyfluthrin (0.056 kg [AI]/ha) for control of cotton bollworm, Helicoverpa zea (Boddie), provided effective control of pentatomids in NuCOTN33B or conventional 'DP5415' and increased yields compared with untreated plots. Fiber quality did not differ among treated or untreated plots of NuCOTN33B. The ground-cloth technique was used to estimate populations of stink bugs, and data indicated that treatment at one bug per 2 m of row adequately protected cotton from yield loss due to stink bug damage. Observations on boll damage indicated that treatment might be necessary if >20-25% reveal internal symptoms of feeding injury during mid- to late season. More detailed damage thresholds should be developed to complement an approach based on population monitoring. This study validated current recommendations for management of pentatomids in cotton, demonstrated the necessity of threshold use for stink bugs in transgenic cultivars expressing endotoxin from B. thuringiensis, and provided insight into further development of management options for pentatomids in the crop.     

Inter- and intraspecific variation in defensive compounds produced by five neotropical stink bug species (Hemiptera: Pentatomidae)

The differences in composition of defensive secretions between nymphs, adult males and adult females of Chinavia impicticornis (=Acrosternum impicticorne), Chinavia ubica (=Acrosternum ubicum), Euschistus heros, Dichelops melacanthus and Piezodorus guildinii (Hemiptera, Pentatomidae) were analysed within and between species using compositional log-ratio statistics and canonical variates analysis. Differences in composition between nymphs, males and females were found for all species, as well as when all species were pooled. In particular, tetradecanal appears to be a predominantly nymphal compound in D. melacanthus, E. heros and P. guildinii. In the two Chinavia species 4-oxo-(E)-2-hexenal and an unknown compound were more dominant in nymphs. The interspecific analysis revealed a good separation of defensive compounds according to their taxonomic relationship. Thus, the two Chinavia species grouped together, with (E)-2-decenal and (E)-2-hexenyl acetate, contributing to this separation. The other three species also differed from each other, with (E)-2-octenal associated to D. melacanthus, (E)-2-hexenal to P. guildinii and (E,E)-2,4-decadienal and tetradecanal to E. heros. The pooled analysis of stage ignoring species revealed tetradecanal and 4-oxo-(E)-2-decenal (tentative identification) strongly associated to nymphs. Thus, there are predictable differences between stages, and many of the differences are conserved between species. Consideration of these differences could prove to be important in understanding stink bug-natural enemy interactions, and in optimising biocontrol efforts.     

Mate location in the southern green stink bug,Nezara Viridula (Heteroptera: Pentatomidae), mediated through substrate-borne signals on ivy

We observed mate location behavior in Nezara viridulaon the ivy Hedera helix.The female stays motionless on the plant stem, calling with rhythmic repetitions of her calling song. The male walks on the plant, answering with his calling and courtship songs. On encountering petioles or branching points, the male stops and straddles his legs across the fork. Only after another repetition of the female's call does the male proceed to walk again. This behavior may enable the male to compare the vibrations in the two substrates and choose the stronger one to follow. On plants where visual and airborne acoustic communication can be prevented by leaves, substrateborne vibrational signals perceived by receptors in legs would enhance the likelihood of mate location in N. viridula.     

Field attraction of the stink bug Euschistus conspersus (Hemiptera: Pentatomidae) to synthetic pheromone-baited host plants.

The attraction of the stink bug Euschistus conspersus Uhler to sources of the synthetic pheromone component methyl (2E,4Z)-decadienoate was investigated in a series of field experiments in native vegetation surrounding commercial apple orchards in Washington. In experiments with pheromone lures placed inside two different tube-type traps, stink bugs were attracted to the immediate area around traps in large numbers, but very few were caught in the traps. Pheromone lures attached directly to the host plant mullein, Verbascum thapsus L., demonstrated that these 'baited" plants attracted significantly more E. conspersus than unbaited plants. Spring (reproductive) and summer (reproductively diapausing) E. conspersus adults, both males and females, were attracted to pheromone-baited plants. There was no significant difference in the number of male or female E. conspersus attracted to pheromone-baited traps or plants in any of the experiments, further characterizing methyl (2E,4Z)-decadienoate as an aggregation, and not a sex pheromone. Stink bug aggregations formed within 24-48 h of lure placement on mullein plants and remained constant until the lure was removed after which aggregations declined over 3-4 d to the level of unbaited plants. The implications of these studies for E. conspersus monitoring and management are discussed.     

Assessing Nezara viridula (Hemiptera: Pentatomidae) feeding damage in macadamia nuts by using a biological stain

Damage caused by southern green stink bug, Nezara viridula (L.), to macadamia nuts, Macadamia integrifolia Maiden & Betche, is normally determined after nuts are harvested and processed, which may be many months after damage occurred in the field. We developed a method using ruthenium red dye to stain stink bug feeding probes and indirectly assess feeding activity in macadamia nuts. By using the staining method, feeding probes were easily detected on the husk, shell, and kernel. Husk probing was highly correlated (0.80-0.90) with feeding and damage to the kernel. Failure rate to detect kernel damage from stained husk probes was generally <6%. The staining method was equally effective for immature and mature nuts; therefore, N. viridula feeding activity can be monitored throughout the season to evaluate pest management tactics and forecast outbreak populations.     

Influence of non‐crop plants on stink bug (Hemiptera: Pentatomidae) and natural enemy abundance in tomatoes

We investigated the effects of weed hosts on stink bug density and damage (Euschistus conspersus Uhler and Thyanta pallidovirens Stal), and a nectar bearing plant on natural enemies of stink bugs in the Sacramento Valley of California. Stink bug density and fruit damage were evaluated in processing tomatoes adjacent to weedy and cultivated borders. The density of E. conspersus was significantly greater in tomatoes adjacent to weedy borders in July but not during August/September. Thyanta pallidovirens was less abundant overall (19%), but was found in significantly greater densities adjacent to cultivated borders in July but not in August/September. Mean percent fruit damage by stink bugs was greater adjacent to the weedy border than the cultivated border, but this difference was not significant. Stink bug egg parasitism and generalist predator density were evaluated in fresh market tomatoes adjacent to a sweet alyssum (Lobularia maritima L.) border and an unplanted control border at three sites. Egg parasitism was significantly greater in the alyssum treatment for the 9–12 September sampling period. Jalysus wickhami VanDuzee (Hemiptera: Berytidae) density was significantly greater in the alyssum treatment in mid‐June. No other significant differences in predator populations were detected. Results of these two studies show that habitat manipulations have the potential to reduce densities of E. conspersus in tomato, the first step in developing a farmscape management plan for stink bug control.     

Toxicity of insecticides in a glass-vial bioassay to adult brown, green, and southern green stink bugs (Heteroptera: Pentatomidae)

Adult brown, Euschistus servus (Say); green, Acrosternum hilare (Say); and southern green, Nezara viridula (L.), stink bugs were collected from soybean, Glycine max (L.) Merr., in fall 2001 and 2002 near Stoneville, MS, and Eudora, AR. A glass-vial bioassay was used to determine LC50 values for the three species of stink bugs for the pyrethroids bifenthrin, cypermethrin, cyfluthrin, lambda-cyhalothrin, and permethrin, and the organophosphates acephate, dicrotophos, malathion, and methyl parathion. Results confirmed findings of other researchers that the brown stink bug was less susceptible to pyrethroid and organophosphate insecticides than were green and southern green stink bugs. The susceptibility of all three stink bug species to the insecticides tested was very similar at both test locations. The study established baseline insecticide mortality data from two locations in the mid-South for three stink bug species that are pests of soybean and cotton, Gossypium spp. Data from the tests are valuable for future use in studies on resistance and in resistance monitoring programs.     

A native predator utilising the invasive brown marmorated stink bug,Halyomorpha halys (Hemiptera: Pentatomidae) as a food source

The nest provisions of a native predator species Bicyrtes quadrifasciata (Say) (Hymenoptera: Crabronidae) were surveyed and examined for the presence of nymphs of Halyomorpha halys, which is an invasive pest of many economically important agricultural crops in the U.S.A. We found 96% of the nest provisions of B. quadrifasciata were H. halys indicating the potential of B. quadrifasciata in biocontrol of this pest. Potential of B. quadrifasciata in regulating population dynamics of H. halys is discussed.