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.     

Effect of relative humidity on emergence and on dispersal and regrouping of first instar Nezara viridula (L.) (Hemiptera: Pentatomidae)

Laboratory studies with 1st instar of southern green stink bug, Nezara viridula (L.) indicated that relative humidity (RH) greatly affected nymph emergence and survivorship up to the 2nd instar, reaching the maximum value (approximately 90%) with RH of > 80%. At 60% RH, 60% of the nymphs emerged and survived, while with 0% RH only approximately 15% of eggs hatched, and most nymphs died. Emerged nymphs from egg masses placed in plastic boxes with a gradient of humidity remained on egg shells for ca. one day. After this period, they dispersed and regrouped on top of shells 6.8 +/- 0.67 times, until they abandoned the shells toward the source of humidity, avoiding the water-saturated areas. Duration taken for each rearrangement (dispersal + regroup) increased with time, with a range of approximately 26 min to 44 min. The mean duration of the grouping behavior on egg shells after each rearrangement decreased from approximately 102 min (1st) to 24 min (6th and last grouping). The rearrangement behavior of 1st instars on top of egg shells apparently compensates for the water loss of nymphs.     

The Effect of Seasonal Changes on Nezara viridula (L.) (Hemiptera: Pentatomidae) and Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) in Hawaii

The effect of seasonal changes in temperature and photoperiod on the interaction of Nezara viridula (Hemiptera: Pentatomidae) and its egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae) was investigated in the laboratory. We found no evidence of reproductive diapause in N. viridula under simulated Hawaiian summer and winter conditions. Further, although “diapause” coloration was obtained in the laboratory, it was not correlated with reproductive status. Studies of the survival of T. basalis provided with honey under the same simulated conditions showed that under summer conditions, only 2.1% of the female and 13.5% of the male population were still alive by 60 days. When provided with N. viridula egg masses at 30 days, 79.4% of the eggs were parasitized in a 3-h period. Under winter conditions, 54.3, 28.3, and 14.5% of the females were alive at 30, 60, and 90 days after adult emergence. When provided with N. viridula egg masses at 30, 60, and 90 days for 3 h, 57.6, 32.8, and 47.1% of the eggs were successfully parasitized. These studies suggest the limiting factor in the interaction of T. basalis and N. viridula is not reproductive diapause, but instead the ability of T. basalis to survive summer conditions.     

Geographical distribution of genetically determined types of Nezara viridula (L.) (Heteroptera: Pentatomidae) in Brazil

Nezara viridula (L.) is a cosmopolitan and polymorphic pentatomid. Several genetically determined types have been described due to body color variation in adults. A survey covering 13 Brazilian states was conducted during 2001 and 2002 to determine the geographical distribution of the main types. Type G (smaragdula--body entirely green), the most common, showed a wide distribution, from south to north (latitude 2 degrees 49' N to 31 degrees 46' S), except in the Central-West Region. Type O (torquata--body green with lateral and median lobes of the head and anterior margin of the pronotum yellow), less abundant than the former, was more frequent at latitudes > 23 degrees 18' S and mean annual temperatures < 20.8 degrees C (Southern Region). Type Y (aurantiaca--body entirely gold or orange), which is rare, was collected only in the Southern Region. N. viridula (smaragdula) was captured in only one place in the Northern Region (Boa Vista, RR, latitude 2 degree 49' N). The abundance of the two most common types, smaragdula and torquata, was not correlated with altitude.     

Potential use of antibiotic to improve performance of laboratory-reared Nezara viridula (L.) (Heteroptera: Pentatomidae)

The antibiotic streptomycin added to the drinking water at a concentration of 125 mg/ l during nymphal development of Nezara viridula (L.) (Heteroptera: Pentatomidae)accelerated the development in ca. 2 days, increased survivorship, and doubled adult longevity; nymph survivorship and adult body weight were not affected when compared to control insects. Streptomycin has potential in rearing N. viridula, especially in improving quality of field-collected adults, by mitigating the introduction of pathogenic bacteria, and improving the quality of the population.     

Influence of azadirachtin on development and reproduction of Nezara viridula L. (Het., Pentatomidae)

Abstract:  The potential of azadirachtin as an insect growth regulator was evaluated in Nezara viridula L. by applying different doses to fifth instar nymphs. At doses of 200–500 ng per insect almost all the individuals died during moulting to adults and the survivors showed a great number of nymphal characteristics, and also died very soon after. At lower doses, from 2 to 50 ng per insect, approximately 50% of the adults seemed normal. No effect was observed during the last nymphal instar, even at the highest dose applied. The fertility and fecundity of adults, males and females, developing from nymphs treated with different low doses of azadirachtin were also studied. When azadirachtin was applied at 20 ng per insect on fifth instar nymphs, females developing from these nymphs showed a conspicuous reduction of fecundity. This same effect was evident when untreated females mated with males obtained from treated nymphs. Finally, azadirachtin applied at 1  μ g/cm² to the oviposition surface showed a remarkable oviposition repellency.     

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.     

Feeding preference and movement of Nezara viridula and Euschistus serous (Hemiptera: Pentatomidae) on individual cotton plants

Experiments were conducted in an environmental growth chamber to determine the movement and feeding preferences of Nezara viridula (L.) and Euschistus serous (Say) on individual cotton plants. Fifth instars were caged by species on a single cotton plant (FM 9063 B2F) containing four discrete boll sizes ranging from 1.1 to 3.0 cm in diameter over a period of 5 d per replication. Two digital video cameras were simultaneously focused on each of the four bolls per plant to visually confirm stink bug resting and movement. During the study, a total of 4,080 h of video footage was recorded and analyzed. Results showed that N. viridula and E. serous did not prefer the exact same boll sizes. In a trial with eight stink bugs per plant, N. viridula spent more time on the three larger boll classes, 1.6-2.0, 2.1-2.5, and 2.6-3.0 cm. In a separate trial with one stink bug per plant, N. viridula spent more time on the larger boll classes while E. serous exhibited the strongest preference for 1.1-1.5 and 2.1-2.5 cm bolls. N. viridula moved more often than E. serous and both species moved more often during photophase compared with scotophase. Regardless of species or number of bugs released, bolls in the smallest boll size class fell off the plant about 3 d after the bugs were released. These results confirm that scouts who are estimating stink bug damage should select bolls in the 2.1-2.5 cm diameter boll size class.     

Development of a dry artificial diet for Nezara viridula (L.) and Euschistus heros (Fabricius) (Heteroptera: Pentatomidae)

Artificial diets prepared with wheat germ, soybean protein, dextrosol, potato starch, sucrose, cellulose, soybean or sunflower oil, and vitamin solution for rearing Nezara viridula (L.) and Euschistus heros (Fabricius) were tested under controlled temperature (25 +/- 1 degrees C), RH (60 +/- 10%), and photophase (14h). Three diets were tested and compared with the natural diet privet [soybean and peanut seeds and privet Ligustrum lucidum Ait. fruit (Oleaceae)]. All three artificial diets allowed full development. The diet containing sunflower oil was the most suitable for N. viridula while E. heros developed better on a diet composed of soybean oil. Data indicated that the artificial diets were inferior to the natural diet. The artificial diets were more adequate for E. heros.     

Variation in mitochondrial cytochrome c oxidase subunit I gene in Nezara viridula (Hemiptera: Pentatomidae) from Argentina

Here, we examine the genetic diversity in the agricultural pest Nezara viridula (Linnaeus, 1758 ) from populations of Argentina using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. The DNA sequence comparisons of 718 base pairs of the COI gene revealed seven haplotypes. The observed total haplotype diversity (Hd) value was of 0.138, and the nucleotide diversity was of 0.00039 and 0.00135 according to π and θ_W, respectively. Eight out of the 10 populations analysed, mostly from soya bean crops, only presented the more frequent haplotype, while 2 haplotypes were found in a mixed culture and 6 haplotypes in a peanut culture. Factors such as differential insecticide applications, as well as the surrounding habitat, and the host plant preference could be related to the genetic diversity differences observed among samples of N. viridula. The analysis of genetic diversity in samples collected in crops treated and non‐treated with insecticides, as well as in samples collected from different seasons, could help to clarify the role of the factors that led to the pattern of genetic diversity detected in this study. The result of a comparative analysis of COI gene sequences among populations from South America, Africa, Asia and Europe was consistent with the hypothesis of an African origin of N. viridula. On the other hand, the haplotypes of Europe were clustered with haplotypes from South America. In addition, specimens from Madeira (west of Europe) shared ancestry with South America and Europe. It has been suggested that a probable route of colonization of America could have been from Western Europe towards the eastern coasts of South America.     

Susceptibility of Nezara viridula (L.) (Hemiptera: Pentatomidae) Egg Masses of Different Sizes to Parasitism by Trissolcus basalis (Woll.) (Hymenoptera: Platygastridae) in the Field

Egg masses of Nezara viridula (L.) are commonly parasitized by Trissolcus basalis (Woll.), and we investigated the role of size of egg masses on parasitization by T. basalis. Sentinel egg masses were exposed to parasitism in the field for 6–7 days, when they were collected for evaluation of parasitoid emergence. We recorded the number of eggs per egg mass, the number of emerged hosts, and the number of empty and parasitized eggs. We calculated the proportion of attacked host egg masses (DE), the proportion of parasitized eggs per attacked egg mass (PE), and total parasitism (PI). The total number of egg masses exposed to parasitism was 330. The minimum, mean, and maximum egg mass sizes were 25, 75.2, and 111, respectively. DE and PE varied widely between different fields, and they were independent of egg mass size. In 14.2% of all parasitized egg masses, we found simultaneous emergence of T. basalis and N. viridula independently of host egg mass size. PE exhibited low variability compared with PI and DE, which were linearly related. PI and DE values from other field studies are consistent with the linear relationship, suggesting that PI is mostly related to the proportion of the DE. This also suggests that total parasitism is independent of egg mass size, of possible differences in plant species, and T. basalis density and strains.     

Temporal processing of vibratory communication signals at the level of ascending interneurons in Nezara viridula (Hemiptera: Pentatomidae)

During mating, males and females of N. viridula (Heteroptera: Pentatomidae) produce sex- and species-specific calling and courtship substrate-borne vibratory signals, grouped into songs. Recognition and localization of these signals are fundamental for successful mating. The recognition is mainly based on the temporal pattern, i.e. the amplitude modulation, while the frequency spectrum of the signals usually only plays a minor role. We examined the temporal selectivity for vibratory signals in four types of ascending vibratory interneurons in N. viridula. Using intracellular recording and labelling technique, we analyzed the neurons' responses to 30 pulse duration/interval duration (PD/ID) combinations. Two response arrays were created for each neuron type, showing the intensity of the responses either as time-averaged spike counts or as peak instantaneous spike rates. The mean spike rate response arrays showed preference of the neurons for short PDs (below 600 ms) and no selectivity towards interval duration; while the peak spike rate response arrays exhibited either short PD/long ID selectivity or no selectivity at all. The long PD/short ID combinations elicited the weakest responses in all neurons tested. No response arrays showed the receiver preference for either constant period or duty cycle. The vibratory song pattern selectivity matched the PD of N. viridula male vibratory signals, thus pointing to temporal filtering for the conspecific vibratory signals already at level of the ascending interneurons. In some neurons the responses elicited by the vibratory stimuli were followed by distinct, regular oscillations of the membrane potential. The distance between the oscillation peaks matched the temporal structure of the male calling song, indicating a possible resonance based mechanism for signal recognition.     

Trap cropping to manage green vegetable bug Nezara viridula (L.) (Heteroptera: Pentatomidae) in sweet corn in New Zealand

Abstract

• 1 The use of trap crops to reduce green vegetable bug (GVB) Nezara viridula (L.) (Heteroptera: Pentatomidae) damage to process sweet corn Zea mays (L.) was investigated in three field experiments.
• 2 In the first season, small plots (2.7 m by 10 m) of white mustard Sinapis alba (L.) with pea Pisum sativum (L.) were sown along a crop border and compared with sweet corn alone.
• 3 In the second season, black mustard Brassica nigra (L.) was sown at two sowing dates (14 days apart) and compared with a sweet corn control, to examine how the maturity of the trap crop affected numbers of GVB trapped.
• 4 A field scale experiment was also conducted to determine the effectiveness of black mustard as a trap crop to protect larger areas of sweet corn from GVB.
• 5 In all three experiments, GVB populations were much higher on the trap crops compared with the sweet corn.
• 6 In both small plot experiments, GVB were contained largely within the trap crop for 2 weeks until the sweet corn was harvested.
• 7 The field scale experiment demonstrated the efficacy of the trap cropping technique to protect larger areas of crop from GVB.
• 8 Percentages of damaged sweet corn cobs in the outside row of fields protected by a trap crop were 0% and 1%, respectively, compared with 11% and 22% in control fields.
• 9 Trap cropping is recommended as an effective strategy to manage this insect. Options for cultivating or spraying the trap crops to reduce bug survival are discussed.

     

Genetic differentiation of geographically separated populations of the southern green stink bug Nezara viridula (Hemiptera: Pentatomidae)

Genetic variation in the southern green stink bug Nezara viridula (Linnaeus) from 11 geographically separated sampling locations (Slovenia, France, Greece, Italy, Madeira, Japan, Guadeloupe, Galapagos, California, Brazil and Botswana) was studied by sequencing 16S and 28S rDNA, cytochrome b and cytochrome c oxidase subunit I gene fragments and random amplified polymorphic DNA (RAPD) analysis. Sequencing revealed 11 distinct haplotypes clustering into lineages A, B and C. Lineage C was characteristic for a single analysed specimen from Botswana. Lineage B was detected in Japan, and it probably arose in Asia. Haplotypes of European and American specimens belonged to lineage A; specimens from France, Slovenia, Madeira and Brazil shared highly similar haplotypes (>99%) from subgroup A1, while all the specimens from Greece, California, Galapagos and Guadeloupe shared a haplotype from subgroup A2. RAPD data were more variable but consistent with mtDNA sequences, revealing the same clustering. They separated the Botswanian specimen from Japanese specimens and from a group of more closely related specimens from Europe and America. Sequence and RAPD results both support the African origin of N. viridula, followed by dispersal to Asia (lineage B) and, more recently, by expansion to Europe and America (lineage A). RAPD analysis revealed two highly supported subgroups in Japan, congruent with mtDNA lineages A2 and B, suggesting multiple colonization of Japan. Invariant sequences at the 28S rDNA combined with other results do not support the hypothesis that cryptic (sibling) species exist within the populations investigated in this study.     

Application of neem seed kernel extracts result in mouthpart deformities and subsequent mortality in Nezara viridula (L.) (Hem: Pentatomidae)

Abstract:  The effect of topical application of neem seed kernel aqueous suspension (NSKAS) and neem seed kernel hexane extract (NSKHE) on 2-day-old fourth instar nymphs of Nezara viridula (L.) was studied under laboratory conditions. The applications resulted in loosening of the proboscis, along with separation and deformation of mouthparts, predominantly in the stylets in fifth instar nymphs, adults and in inter-stadial moults. This rendered the insects incapable of feeding and led them to death. Application of NSKAS at 2.5% concentration led to 100% mortality, of which 40% was due to mouthpart deformities. Application of NSKHE at 2.5% concentration led to only 70% mortality, of which 3.33% was due to mouthpart deformities. At 5% concentration, mortality was again 100% for NSKAS and reached close to 100% for the NSKHE application. Dose-dependent mortality was observed for both the applications. However, dose-dependent mouthpart deformities were observed only for NSKAS, the highest being 60% at the 5% concentration. Moreover, for both the applications, more than 50% of the total mortality was observed up to the fifth instar at almost all the concentrations. The LC₅₀ values for NSKAS and NSKHE applications, for mortality up to the fifth instar were 0.36% and 5.22%, and for mortality to the adult stage (10 days old) they were 0.23% and 1.32% respectively. The percentage of bugs showing mouthpart deformities and toxicity was higher for NSKAS than NSKHE, and indicated that NSKAS was more potent than NSKHE.     

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.