Transcriptional profiling reveals a critical role of GmFT2a in soybean staygreen syndrome caused by the pest Riptortus pedestris.

Soybean staygreen syndrome, characterized by delayed leaf and stem senescence, abnormal pods, and aborted seeds, has recently become a serious and prominent problem in soybean production. Although the pest Riptortus pedestris has received increasing attention as the possible cause of staygreen syndrome, the mechanism remains unknown. Here, we clarify that direct feeding by R. pedestris, not transmission of a pathogen by this pest, is the primary cause of typical soybean staygreen syndrome and that critical feeding damage occurs at the early pod stage. Transcriptome profiling of soybean indicated that many signal transduction pathways, including photoperiod, hormone, defense response, and photosynthesis, respond to R. pedestris infestation. Importantly, we discovered that members of the FLOWERING LOCUS T (FT) gene family were suppressed by R. pedestris infestation, and overexpression of floral inducer GmFT2a attenuates staygreen symptoms by mediating soybean defense response and photosynthesis. Together, our findings systematically illustrate the association between pest infestation and soybean staygreen syndrome and provide the basis for establishing a targeted soybean pest prevention and control system.

Irradiation-induced sterility in an egg parasitoid and possible implications for the use of biological control in insect eradication.

Classical biological control is a pest control tool involving the release of imported natural enemies. The Sterile Insect Technique (SIT) comprises releasing sexually sterile insects of a pest into the wild population for suppression or eradication. Both these approaches are environmentally friendly and their combination can result in a synergistic impact on pest populations and improve eradication. However, stringent regulation surrounding the introduction of biological control agents limits their use in eradication owing to the perceived risk of effects on non-target organisms. We investigated the irradiation biology of the egg parasitoid Trissolcus basalis to ascertain whether sterile parasitoids could mitigate the risk of potential sustained non-target impacts. Mated female T. basalis were gamma-irradiated at doses between 120 and 150 Gy and exposed to egg masses of their host Nezara viridula throughout their lifespans. This resulted in host mortality, despite a substantial reduction in developing parasitoid offspring, which followed a negative dose-response. There was no emergence of parasitoid offspring at 140 Gy and above. Irradiation did not affect oviposition behaviour but caused an increase in longevity. Consequently, sterile parasitoids could possibly alleviate concerns regarding the irreversibility of biological control release, which promotes further investigation of their potential role in eradication.

Sublethal and transgenerational effects of sulfoxaflor on the demography and feeding behaviour of the mirid bug Apolygus lucorum.

Sulfoxaflor, the first commercially available sulfoximine insecticide, has been used for the control of sap-feeding insect pests such as plant bugs and aphids on a variety of crops. However, its sublethal effects on the mirid bug Apolygus lucorum, one of the key insect pests of Bt cotton and fruit trees in China, have not been fully examined. Here, we evaluated the demography and feeding behaviour of A. lucorum exposed to sulfoxaflor. The leaf-dipping bioassay showed that the LC10 and LC30 of sulfoxaflor against 3rd-instar nymphs of this insect were 1.23 and 8.37 mg L-1, respectively. The LC10 significantly extended the nymphal duration and decreased the oviposition period by 5.29 days and female fecundity by 56.99% in the parent generation (F0). The longer duration of egg, 5th-instar nymphs, preadult, and male adult longevity were observed in the F1 generation (F1) at LC10. At the LC30, the duration of egg and 1st-instar nymph, female adult longevity, and oviposition period of the F1 were significantly shorter, while the nymphal duration in the F0 and duration of 5th-instar nymphs, preadult survival rate, and male adult longevity in the F1 significantly increased. The net reproductive rate (R0), intrinsic rate of increase (r), and finite rate of increase (λ) in the F1 were not significantly affected by these two concentrations, whereas the mean generation time (T) was lower at the LC30. Additionally, the probe counts and cells mixture feeding time were markedly lengthened by the LC10 and LC30, respectively, when A. lucorum nymphs exposed to sulfoxaflor fed on Bt cotton plants without insecticides. These results clearly indicate that sulfoxaflor causes sublethal effects on A. lucorum and the transgenerational effects depend on the tested concentrations.

Salicylic acid induced by herbivore feeding antagonizes jasmonic acid mediated plant defenses against insect attack.

We recently reported the transcriptomic signature of salicylic acid (SA) and jasmonic acid (JA) biosynthetic and responsive genes in Arabidopsis thaliana plants infested with the herbivore Eurydema oleracea. We demonstrated that insect feeding causes induction of both SA- and JA-mediated signaling pathways. Using transgenic SA-deficient NahG plants, we also showed antagonistic cross-talk between these two phytohormones. To gain more insight into the roles of the SA and JA pathways in plant defenses against E. oleracea, we report here on the dynamics of SA and JA levels in the wild-type genotype Col-0 and the transgenic Arabidopsis NahG mutant that does not accumulate SA. We show that SA strongly accumulates in the wild-type plants after 24 h of herbivore infestation, while JA levels do not change significantly. On the contrary, in the infested NahG plants, SA levels were not affected by E. oleracea feeding, whereas JA levels which were constitutively higher than the wild-type did not significantly change after 6 hours of herbivore feeding. Accordingly, when the wild-type and the jar1-1 mutant (which fails to accumulate JA-Ile) Arabidopsis plants were challenged with E. oleracea in a two-choice arena, the insect fed preferentially on the jar1-1 plants over the wild-type. These data support the conclusion that E. oleracea infestation strongly induces the SA pathway in the wild-type, thus antagonizing JA-mediated plant defenses against herbivory, as a strategy to suppress plant immunity.

Patterns of Genome-Wide Variation, Population Differentiation and SNP Discovery of the Red Banded Stink Bug (Piezodorus guildinii).

Unravelling the details of range expansion and ecological dominance shifts of insect pests has been challenging due to the lack of basic knowledge about population structure, gene flow, and most importantly, how natural selection is affecting the adaptive process. Piezodous guildinii is an emerging pest of soybean in the southern region of the United States, and increasingly important in Brazil in recent years. However, the reasons P. guildinii is gradually becoming more of a problem are questions still mostly unanswered. Here, we have genotyped P. guildinii samples and discovered 1,337 loci containing 4,083 variant sites SNPs that were used to estimate genetic structure and to identify gene candidates under natural selection. Our results revealed the existence of a significant genetic structure separating populations according to their broad geographic origin, i.e., U.S. and Brazil, supported by AMOVA (FGT = 0.26), STRUCTURE, PCA, and FST analyses. High levels of gene flow or coancestry within groups (i.e., within countries) can be inferred from the data, and no spatial pattern was apparent at the finer scale in Brazil. Samples from different seasons show more heterogeneous compositions suggesting mixed ancestry and a more complex dynamic. Lastly, we were able to detect and successfully annotated 123 GBS loci (10.5%) under positive selection. The gene ontology (GO) analysis implicated candidate genes under selection with genome reorganization, neuropeptides, and energy mobilization. We discuss how these findings could be related to recent outbreaks and suggest how new efforts directed to better understand P. guildinii population dynamics.

Bioactivity of the Cymbopogon citratus (Poaceae) essential oil and its terpenoid constituents on the predatory bug, Podisus nigrispinus (Heteroptera: Pentatomidae).

Podisus nigrispinus Dallas (Heteroptera: Pentatomidae), released in biological control programs, is a predator of Lepidopteran and Coleopteran species. Lemongrass essential oil and its constituents can be toxic to this natural enemy. The major constituents of lemongrass essential oil are neral (31.5%), citral (26.1%), and geranyl acetate (2.27%). Six concentrations of lemongrass essential oil and of its citral and geranyl acetate constituents were applied to the thorax of P. nigrispinus nymphs and adults. The walking and respiratory behavior of the P. nigrispinus third-instar nymphs, treated with citral and geranyl acetate at the LD50 and LD90 doses, were analyzed with video and respirometer. The lemongrass essential oil toxicity increased from first- to fifth-instar P. nigrispinus nymphs. The P. nigrispinus respiration rates (µL de CO2 h-1/insect) with citral and geranyl acetate in the LD50 and LD90 differed. Nymphs exposed to the lemongrass essential oil and its constituents on treated surfaces presented irritability or were repelled. Podisus nigrispinus adults were tolerant to the lemongrass essential oil and its constituents, geranyl acetate and citral. The altered respiratory activity with geranyl acetate and the fact that they were irritated and repelled by citral suggest caution with regard to the use of the lemongrass essential oil and its constituents in integrated pest management incorporating this predator, in order to avoid diminishing its efficiency against the pests.

Lethal and behavioral effects of synthetic and organic insecticides on Spodoptera exigua and its predator Podisus maculiventris.

BACKGROUND: The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is a key insect pest of edible vegetables around the world and it is resistant to insecticide of different classes. Insecticides that are effective to this pest and selective to predator stinkbugs are required for the integrated management of S. exigua. METHODS: The toxicity of four commercial insecticide formulations azadirachtin + pyrethrin, spinosad, pyrethrin and chlorantraniliprole was tested on the target pest and their side effect were evaluated on the spined soldier bug, Podisus maculiventris (Heteroptera: Pentatomidae) through different bioassays. RESULTS: Spinosad and chlorantraniliprole were more toxic to S. exigua than to the predator P. maculiventris but opposite results were obtained for pyrethrin and azadirachtin + pyrethrin in contact toxicity bioassay. Chlorantraniliprole was the most toxic to S. exigua in oral toxicity bioassay, followed by spinosad, pyrethrin and azadirachtin + pyrethrin. Spinosad in oral toxicity bioassay was the most toxic to P. maculiventris, followed by pyrethrin, azadirachtin + pyrethrin and chlorantraniliprole. Spinosad caused irritability to the predator while pyrethrin to the pest. The insecticide repellency was not observed over the tested insect species. The synthetic insecticide chlorantraniliprole was less toxic than the natural pyrethrin, azadirachtin + pyrethrin and spinosad to the predator. CONCLUSIONS: This work provides useful information on the combination of commercial insecticides with the predator P. maculiventris to controlling S. exigua in integrated pest management (IPM) programs.

Characterized non-transient microbiota from stinkbug (Nezara viridula) midgut deactivates soybean chemical defenses.

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 midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro ß-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses.

The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields.

The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.

Characterizing the spatial distribution of brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), populations in peach orchards.

Geospatial analyses were used to investigate the spatial distribution of populations of Halyomorpha halys, an important invasive agricultural pest in mid-Atlantic peach orchards. This spatial analysis will improve efficiency by allowing growers and farm managers to predict insect arrangement and target management strategies. Data on the presence of H. halys were collected from five peach orchards at four farms in New Jersey from 2012-2014 located in different land-use contexts. A point pattern analysis, using Ripley's K function, was used to describe clustering of H. halys. In addition, the clustering of damage indicative of H. halys feeding was described. With low populations early in the growing season, H. halys did not exhibit signs of clustering in the orchards at most distances. At sites with low populations throughout the season, clustering was not apparent. However, later in the season, high infestation levels led to more evident clustering of H. halys. Damage, although present throughout the entire orchard, was found at low levels. When looking at trees with greater than 10% fruit damage, damage was shown to cluster in orchards. The Moran's I statistic showed that spatial autocorrelation of H. halys was present within the orchards on the August sample dates, in relation to both populations density and levels of damage. Kriging the abundance of H. halys and the severity of damage to peaches revealed that the estimations of these are generally found in the same region of the orchards. This information on the clustering of H. halys populations will be useful to help predict presence of insects for use in management or scouting programs.

Double strand RNA delivery system for plant-sap-feeding insects.

Double-stranded RNA (dsRNA)-mediated gene silencing, also known as RNA interference (RNAi), has been a breakthrough technology for functional genomic studies and represents a potential tool for the management of insect pests. Since the inception of RNAi numerous studies documented successful introduction of exogenously synthesized dsRNA or siRNA into an organism triggering highly efficient gene silencing through the degradation of endogenous RNA homologous to the presented siRNA. Managing hemipteran insect pests, especially Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is critical to food productivity. BMSB was recently introduced into North America where it is both an invasive agricultural pest of high value specialty, row, and staple crops, as well as an indoor nuisance pest. RNAi technology may serve as a viable tool to manage this voracious pest, but delivery of dsRNA to piercing-sucking insects has posed a tremendous challenge. Effective and practical use of RNAi as molecular biopesticides for biocontrol of insects like BMSB in the environment requires that dsRNAs be delivered in vivo through ingestion. Therefore, the key challenge for molecular biologists in developing insect-specific molecular biopesticides is to find effective and reliable methods for practical delivery of stable dsRNAs such as through oral ingestion. Here demonstrated is a reliable delivery system of effective insect-specific dsRNAs through oral feeding through a new delivery system to induce a significant decrease in expression of targeted genes such as JHAMT and Vg. This state-of-the-art delivery method overcomes environmental delivery challenges so that RNAi is induced through insect-specific dsRNAs orally delivered to hemipteran and other insect pests.

Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula.

Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest.

Preference and prey switching in a generalist predator attacking local and invasive alien pests.

Invasive pest species may strongly affect biotic interactions in agro-ecosystems. The ability of generalist predators to prey on new invasive pests may result in drastic changes in the population dynamics of local pest species owing to predator-mediated indirect interactions among prey. On a short time scale, the nature and strength of such indirect interactions depend largely on preferences between prey and on predator behavior patterns. Under laboratory conditions we evaluated the prey preference of the generalist predator Macrolophus pygmaeus Rambur (Heteroptera: Miridae) when it encounters simultaneously the local tomato pest Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and the invasive alien pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). We tested various ratios of local vs. alien prey numbers, measuring switching by the predator from one prey to the other, and assessing what conditions (e.g. prey species abundance and prey development stage) may favor such prey switching. The total predation activity of M. pygmaeus was affected by the presence of T. absoluta in the prey complex with an opposite effect when comparing adult and juvenile predators. The predator showed similar preference toward T. absoluta eggs and B. tabaci nymphs, but T. absoluta larvae were clearly less attacked. However, prey preference strongly depended on prey relative abundance with a disproportionately high predation on the most abundant prey and disproportionately low predation on the rarest prey. Together with the findings of a recent companion study (Bompard et al. 2013, Population Ecology), the insight obtained on M. pygmaeus prey switching may be useful for Integrated Pest Management in tomato crops, notably for optimal simultaneous management of B. tabaci and T. absoluta, which very frequently co-occur on tomato.

Paralytic activity of lysophosphatidylcholine from saliva of the waterbug Belostoma anurum.

Lysophosphatidylcholine (LPC) is a major bioactive lipid that is enzymatically generated by phospholipase A(2) (PLA(2)). Previously, we showed that LPC is present in the saliva of the blood-sucking hemipteran Rhodnius prolixus and modulates cell-signaling pathways involved in vascular biology, which aids blood feeding. Here, we show that the saliva of the predator insect Belostoma anurum contains a large number of lipids with LPC accounting for 25% of the total phospholipids. A PLA(2) enzyme likely to be involved in LPC generation was characterized. The activity of this enzyme is 5-fold higher in Belostoma saliva than in other studied hemipterans, suggesting a close association with the predator feeding habits of this insect. Belostoma employs extra-oral digestion, which allows for ingestion of larger prey than itself, including small vertebrates such as amphibians and fish. Therefore, prey immobilization during digestion is essential, and we show here that Belostoma saliva and B. anurum saliva purified LPC have paralytic activity in zebrafish. This is the first evidence that lysophospholipids might play an important role in prey immobilization, in addition to contributing to blood feeding, and might have been an evolutionary acquisition that occurred long before the appearance of hematophagy in this animal group.

Differential responses of forage pearl millet genotypes to chinch bug (Heteroptera: Blissidae) feeding.

Chinch bug, Blissus leucopterus leucopterus (Say) (Heteroptera: Blissidae), is one of the most important insect pests on forage pearl millet, Pennisetum glaucum L. R. Br., production in the southeastern United States. Twenty-nine forage pearl millet genotypes were assessed for chinch bug resistance by using stunt and necrosis ratings in combination with quantitative measurements of chlorophyll content and leaf photosynthetic rate. Plant stunt and leaf sheath necrosis ratings, and chlorophyll content in flag leaves differed among the 29 genotypes. Photosynthetic rate differed both among the noninfested control and among the chinch bug-infested plants. The chinch bug-infested plants had lower photosynthetic rate than the noninfested control plants. Inbreds with resistance superior to that of Tift 23DB were identified for hybrid development. When the 29 pearl millet genotypes were assessed by the six parameters by using cluster analysis, genotypes 07F-1226, 07F-1229, 07F-1231, 07F-1235, 07F-1238, 07F-1239, and 07F-1240 were the most resistant, whereas the genotypes 07F-1220, 07F-1221, 07F-1225, 07F-1227, 07F-1232, 07F-1246, and Tift 23DB were the most susceptible to chinch bug feeding. The rest of the genotypes expressed intermediate responses to the six parameters. To differentiate the physiological impact of chinch bug feeding on light and dark reactions of plant photosynthesis, photosynthesis capacity was assessed using light and CO2 (A/Ci) response curves on noninfested and chinch bug-infested plants of genotypes 07F-1246, 07F-1223, and 07F-1245, which expressed low, intermediate, and high chlorophyll content, respectively. Based on the A/Ci curves, photosynthesis capacity of injured leaves was suppressed in 07F-1223 and 07F-1246, whereas the chinch bug-injured 07F-1245 leaves showed an increase of photosynthetic rate compared with the noninfested plants. In contrast, light response curves were suppressed in the chinch bug-injured plants compared with the noninfested plants of all three genotypes, irrespective of their variations in insect injury ratings. This research demonstrated that visual stunt and necrosis rating methods in combination with chlorophyll and photosynthesis measurements could be used in screening forage pearl millet for chinch bug resistance and deciphering the underlying resistance mechanisms.

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant.

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C68 --> G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C68 --> G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C68 --> G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays.

The piercing-sucking herbivores Lygus hesperus and Nezara viridula induce volatile emissions in plants.

Plant volatiles induced by herbivory are often used as olfactory cues by foraging herbivores and their natural enemies, and thus have potential for control of agricultural pests. Compared to chewing insects and mites, little is known about plant volatile production following herbivory by insects with piercing-sucking mouthparts. Here, we studied factors (insect life stage, gender, the role of salivary glands, and type of bioassay used for volatile induction) that influence the induction of plant volatiles by two agriculturally important hemipterans, Lygus hesperus and Nezara viridula. Feeding on intact cotton by virgin females of L. hesperus induced 2.6-fold greater volatile response compared to that induced by mated females, possibly due to increased feeding activity by virgin females. This plant volatile response was associated with elicitors present in the insect's salivary glands as well as to the degree of mechanical injury. Feeding injury by N. viridula females also increased volatile emissions in intact maize by approximately 2-fold compared to control plants. Maize seedlings injured by N. viridula emitted higher amounts of the monoterpene linalool, the sesquiterpenes (E)-beta-caryophyllene, alpha-trans-bergamotene, and (E,E)-beta-farnesene, and the homoterpene (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, but not amounts of green leaf volatiles, compared to uninjured plants. Emissions from intact maize injured by adult males were lower than those emitted by adult females of the same age and did not differ from those emitted by uninjured plants. Similarly, feeding by virgin female N. viridula followed by excision led to 64% higher quantities of volatiles compared to untreated plants. Volatile emission in excised plants, however, was considerably greater than in intact plants, suggesting that careful consideration must be given to bioassay design in studies of herbivore-induced plant volatiles. Salivary gland extracts of N. viridula led to sesquiterpene emissions approximately 2.5-fold higher than for controls, although no significant differences were observed for green leaf volatiles, monoterpenes, and homoterpenes. These results indicate that L. hesperus and female N. viridula feeding induce volatile production in plants, and that volatile production is affected by gender and life stage of the bug. Although oviposition and mechanical injury by stylets may increase release of volatiles, elicitors from salivary glands of L. hesperus and N. viridula also seem to play a role in the emission of plant volatiles.

Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata.

A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile hornworm larvae. Second, attack from both mirids and hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.

Herbivore-induced plant vaccination. Part II. Array-studies reveal the transience of herbivore-specific transcriptional imprints and a distinct imprint from stress combinations.

Summary Microarray technology has given plant biologists the ability to simultaneously monitor changes in the expression of hundreds of genes, and yet, to date, this technology has not been applied to ecological phenomena. In native tobacco (Nicotiana attenuata), prior attack of sap-feeding mirids (Tupiocoris notatus) results in vaccination of the plant against subsequent attacks by chewing hornworms (Manduca sexta). This vaccination is mediated by a combination of direct and indirect defenses and tolerance responses, which act in concert with the attack preferences of a generalist predator. Here, we use microarrays enriched in herbivore-elicited genes with a principal components analysis (PCA) to characterize transcriptional 'imprints' of single, sequential, or simultaneous attacks by these two main herbivores of N. attenuata. The PCA identified distinctly different imprints left by individual attack from the two species after 24 h, but not after 5 days. Moreover, imprints of sequential or simultaneous attacks differed significantly from those of single attack, suggesting the existence of a distinct gene expression program responsive to the combination of biological stressors. A dissection of the transcriptional imprints revealed responses in direct and indirect defense genes that were well correlated with observed increases in defense metabolites. Attack from both herbivores elicits a switch from growth- to defense-related transcriptional processes, and herbivore-specific changes occur largely in primary metabolism and signaling cascades. PCA of these polygenic transcriptional imprints characterizes the ephemeral changes in the transcriptome that occur during the maturation of ecologically relevant phenotypic responses.