Changes in primary metabolite content may affect thrips feeding preference in soybean crops.

Past research has characterized the induction of plant defenses in response to chewing insect damage. However, little is known about plant responses to piercing-sucking insects that feed on plant cell-contents like thrips (Caliothrips phaseoli). In this study, we used NMR spectroscopy to measure metabolite changes in response to six days of thrips damage from two field-grown soybean cultivars (cv.), known for their different susceptibility to Caliothrips phaseoli. We observed that thrips damage reduces sucrose concentration in both cultivars, while pinitol, the most abundant leaf soluble carbohydrate, is induced in cv. Charata but not in cv. Williams. Thrips did not show preference for leaves where sucrose or pinitol were externally added, at tested concentration. In addition, we also noted that cv. Charata was less naturally colonized and contained higher levels of trigonelline, tyrosine as well as several compounds that we have not yet identified. We have established that preference-feeding clues are not dependent on the plants major soluble carbohydrates but may depend on other types of compounds or leaf physical characteristics.

Lipoxygenases regulate digestive enzyme inhibitor activities in developing seeds of field-grown soybean against the southern green stink bug (Nezara viridula).

Soybean (Glycine max ) is the world's most widely grown seed legume. One of the most important pests that decrease seed quality and reduce yield of soybean crops is the southern green stink bug (Nezara viridula ). Insect damage triggers accumulation of defensive compounds such as protease inhibitors (PIs), isoflavonoids and reactive oxygen species, which are regulated by the lipoxygenase (LOX)-regulated jasmonic acid (JA) to stop insect feeding. This study identified and characterised the role of LOX isoforms in the modulation of chemical defences in seeds of field-grown soybean that decreased digestive enzyme activities of N. viridula after insect attack. Stink bugs attack increased LOX 1 and LOX 2 expression, and activities of LOX 1 and LOX 3 isoenzymes in developing soybean seeds. In addition, stink bug damage and methyl jasmonate application induced expression and activity of both cysteine PIs and trypsin PIs in developing soybean seeds, suggesting that herbivory induced JA in soybean seeds. High PI activity levels in attacked seeds decreased cysteine proteases and α-amylases activities in the gut of stink bugs that fed on field-grown soybean. We demonstrated that LOX isoforms of seeds are concomitantly induced with JA-regulated PIs by stink bugs attack, and these PIs inhibit the activity of insect digestive enzymes. To our knowledge, this is the first study to investigate the participation of LOX in modulating JA-regulated defences against stink bugs in seeds of field-grown soybean, and our results suggest that soybean PIs may inhibit α-amylase activity in the gut of N. viridula .

Confirmation and refutation of very luminous galaxies in the early Universe.

During the first 500 million years of cosmic history, the first stars and galaxies formed, seeding the Universe with heavy elements and eventually reionizing the intergalactic medium1-3. Observations with the James Webb Space Telescope (JWST) have uncovered a surprisingly high abundance of candidates for early star-forming galaxies, with distances (redshifts, z), estimated from multiband photometry, as large as z ≈ 16, far beyond pre-JWST limits4-9. Although such photometric redshifts are generally robust, they can suffer from degeneracies and occasionally catastrophic errors. Spectroscopic measurements are required to validate these sources and to reliably quantify physical properties that can constrain galaxy formation models and cosmology10. Here we present JWST spectroscopy that confirms redshifts for two very luminous galaxies with z > 11, and also demonstrates that another candidate with suggested z ≈ 16 instead has z = 4.9, with an unusual combination of nebular line emission and dust reddening that mimics the colours expected for much more distant objects. These results reinforce evidence for the early, rapid formation of remarkably luminous galaxies while also highlighting the necessity of spectroscopic verification. The large abundance of bright, early galaxies may indicate shortcomings in current galaxy formation models or deviations from physical properties (such as the stellar initial mass function) that are generally believed to hold at later times.

Glyphosate affects larval gut microbiota and metamorphosis of honey bees with differences between rearing procedures.

The honey bee Apis mellifera is a sentinel species of the pollinator community which is exposed to a wide variety of pesticides. In the last half-century, the pesticide most applied worldwide has been the herbicide glyphosate (GLY) used for weed control and with microbiocide effects. After its application in crops, the GLY residues have been detected in flowers visited by honey bees as well as in the stored food of their hives. Therefore, the honey bee brood can ingest the herbicide during larval development. Recent studies proved that GLY has detrimental effects on adult honey bees and other insects associated with the disturbance of their gut microbiota. GLY induces changes in the growth, metabolism and survival of honey bees and stingless bees reared in vitro. However, the effect of GLY on larval microbiota is unknown so far and there are few studies with an in-hive exposure to GLY. For these reasons, this study aims to determine whether GLY induces dysbiosis in honey bee larvae and affects their metamorphosis during the exposure period (pre-defecation) and the post-exposure period. Furthermore, we assessed this herbicide in vitro and in the hive to compare its effects on different rearing procedures. Finally, we tested the pigment BLUE1 as an indirect exposure marker to detect and estimate the in-hive intake concentration of GLY. Our results indicate that the intake of field-relevant concentrations of GLY induced a slowdown in growth with dysbiosis in the larval gut microbiota followed by late effects on their metamorphosis such as teratogenesis and mortality of newly emerged bees. Nevertheless, brood from the same colonies expressed different signs of toxicity depending on the rearing procedure and in a dose-dependent manner.

Chromosomal-level genome assembly of potato tuberworm, Phthorimaea operculella: a pest of solanaceous crops.

The potato tuberworm, Phthorimaea operculella Zeller, is an oligophagous pest feeding on crops mainly belonging to the family Solanaceae. It is one of the most destructive pests of potato worldwide and attacks foliage and tubers in the field and in storage. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis. Here, we report on the genome assembly of P. operculella at the chromosomal level. Using Illumina, Nanopore and Hi-C sequencing, a 648.2 Mb genome was generated from 665 contigs, with an N50 length of 3.2 Mb, and 92.0% (596/648.2 Mb) of the assembly was anchored to 29 chromosomes. In total, 16619 genes were annotated, and 92.4% of BUSCO genes were fully represented. The chromosome-level genome of P. operculella will provide a significant resource for understanding the genetic basis for the biological study of this insect, and for promoting the integrative management of this pest in future.

Feeding on soybean crops changed gut bacteria diversity of the southern green stinkbug (Nezara viridula) and reduced negative effects of some associated bacteria.

BACKGROUND: The southern green stinkbug (Nezara viridula) is a mayor pest of soybean. However, the mechanism underlying stinkbug resistance to soybean defenses is yet ignored. Although gut bacteria could play an essential role in tolerating plant defenses, most studies testing questions related to insect-plant-bacteria interactions have been performed in laboratory condition. Here we performed experiments in laboratory and field conditions with N. viridula and its gut bacteria, studying gut lipid peroxidaxion levels and cysteine activity in infected and unifected nymphs, testing the hypothesis that feeding on field-grown soybean decreases bacterial abundance in stinkbugs. RESULTS: Gut bacterial abundance and infection ratio were higher in N. viridula adults reared in laboratory than in those collected from soybean crops, suggesting that stinkbugs in field conditions may modulate gut bacterial colonization. Manipulating gut microbiota by infecting stinkbugs with Yokenella sp. showed that these bacteria abundance decreased in field conditions, and negatively affected stinkbugs performance and were more aggressive in laboratory rearing than in field conditions. Infected nymphs that fed on soybean pods had lower mortality, higher mass and shorter development period than those reared in the laboratory, and suggested that field conditions helped nymphs to recover from Yokenella sp. infection, despite of increased lipid peroxidation and decreased cysteine proteases activity in nymphs' guts. CONCLUSIONS: Our results demonstrated that feeding on field-grown soybean reduced bacterial abundance and infection in guts of N. viridula and highlighted the importance to test functional activities or pathogenicity of microbes under realistic field conditions prior to establish conclusions on three trophic interactions. © 2022 Society of Chemical Industry.

Proteases inhibitors-insensitive cysteine proteases allow Nezara viridula to feed on growing seeds of field-grown soybean.

The southern green stink bug, Nezara viridula is one of the primary soybean pests and causes significant economic losses around the world. In spite of the high proteases inhibitor (PI) levels, N. viridula can feed on developing seeds of field-grown soybean and reduce crop yields. Although the PI-induced responses have been extensively investigated in many pest insects, there is lack of knowledge about the mechanisms that stink bugs employ to withstand cysteine PIs of soybean seeds. This study demonstrated that feeding on developing seeds of field-grown soybean inhibited total proteases activity of N. viridula, as result of inhibition of cathepsin B-like activity in the gut. In addition, from the 30 digestive cathepsins recognized in this study, 6 were identified as cathepsin B-like. Stink bugs that fed on growing seeds of field-grown soybean had similar gut pH to those reared in the laboratory, and both cathepsin B- and L-like had an optima pH of 6.5. Therefore, using specific proteases inhibitors we found that the main proteolytic activity in the gut is from cysteine proteases when N. viridula feeds on soybean crops. Since cathepsin L-like activity was not inhibited by soybean PIs, our results suggested that N. viridula relays on cathepsin L-like to feed on soybean. To our knowledge no study before has shown the impact of seed PIs of field-grown soybean on digestive proteases (cathepsin B- and L-like) of N. viridula. This study suggests that the activity of PI-insensitive cathepsins L-like in the gut would be part of an adaptive strategy to feed on developing soybean seeds. In agreement, the expansions of cathepsin L-like complement observed in pentatomids could confer to the insects a higher versatility to counteract the effects of different PIs.

Evaluating the Impact of Post-Emergence Weed Control in Honeybee Colonies Located in Different Agricultural Surroundings.

The honeybee Apis mellifera is exposed to agricultural intensification, which leads to an improved reliance upon pesticide use and the reduction of floral diversity. In the present study, we assess the changes in the colony activity and the expression profile of genes involved in xenobiotic detoxification in larvae and adult honeybees from three apiaries located in agricultural environments that differ in their proportion of the crop/wild flora. We evaluated these variables before and after the administration of a mixture of three herbicides during the summer season. The expression of several cytochrome P450 monooxygenases decreased significantly in larvae after post-emergence weed control and showed significant differences between apiaries in the case of honeybee workers. Principal component analysis (PCA) revealed that colonies located in the plot near to a wetland area exhibited a different relative gene expression profile after herbicide application compared with the other plots. Moreover, we found significant positive correlations between pollen collection and the pesticide detoxification genes that discriminated between plots in the PCA. Our results suggest that nutrition may modify herbicide impact on honeybees and that larvae are more harmed than adults in agroecosystems, a factor that will alter the colonies' population growth at the end of the blooming period.

Role of reactive oxygen species and isoflavonoids in soybean resistance to the attack of the southern green stink bug.

Southern green stink bugs (Nezara viridula L.) are one of the major pests in many soybean producing areas. They cause a decrease in yield and affect seed quality by reducing viability and vigor. Alterations have been reported in the oxidative response and in the secondary metabolites in different plant species due to insect damage. However, there is little information available on soybean-stink bug interactions. In this study we compare the response of undamaged and damaged seeds by Nezara viridula in two soybean cultivars, IAC-100 (resistant) and Davis (susceptible), grown under greenhouse conditions. Pod hardness, H2O2 generation, enzyme activities in guaiacol peroxidase (GPOX), catalase (CAT) and superoxide dismutase (SOD) as well as lipoxygenase expression and isoflavonoid production were quantified. Our results showed a greater resistance of IAC-100 to pod penetration, a decrease in peroxide content after stink bug attack, and higher GPOX, CAT and SOD activities in seeds due to the genotype and to the genotype-interaction with the herbivory treatment. Induction of LOX expression in both cultivars and higher production of isoflavonoids in IAC-100 were also detected. It was then concluded that the herbivory stink bug induces pathways related to oxidative stress and to the secondary metabolites in developing seeds of soybean and that differences between cultivars hold promise for a plant breeding program.

Digestive activity and organic compounds of Nezara viridula watery saliva induce defensive soybean seed responses.

The stink bug Nezara viridula is one of the most threatening pests for agriculture in North and South America, and its oral secretion may be responsible for the damage it causes in soybean (Glycine max) crop. The high level of injury to seeds caused by pentatomids is related to their feeding behavior, morphology of mouth parts, and saliva, though information on the specific composition of the oral secretion is scarce. Field studies were conducted to evaluate the biochemical damage produced by herbivory to developing soybean seeds. We measured metabolites and proteins to profile the insect saliva in order to understand the dynamics of soybean-herbivore interactions. We describe the mouth parts of N. viridula and the presence of metabolites, proteins and active enzymes in the watery saliva that could be involved in seed cell wall modification, thus triggering plant defenses against herbivory. We did not detect proteins from bacteria, yeasts, or soybean in the oral secretion after feeding. These results suggest that the digestive activity and organic compounds of watery saliva may elicit a plant self-protection response. This study adds to our understanding of stink bug saliva plasticity and its role in the struggle against soybean defenses.

Author Correction: The use of Leaf Surface Contact Cues During Oviposition Explains Field Preferences in the Willow Sawfly Nematus Oligospilus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The use of Leaf Surface Contact Cues During Oviposition Explains Field Preferences in the Willow Sawfly Nematus Oligospilus.

After an insect herbivore has reached its host plant, contact cues from the leaf surface often determine host acceptance. We studied contact cues during oviposition behavior of a willow pest, the sawfly Nematus oligospilus (Hymenoptera: Tenthredinidae), a specialist feeder on Salix (Salicaceae) trees, and how it determines oviposition preference in lab and field conditions. We described the sequence of behaviors that lead to egg laying on the most and least preferred willow species. Then we studied the morphology of chemosensory structures present on the female antenna, cerci and ovipositor. Since phenolic glycosides (PGs) are the main secondary metabolites present in Salicaceae species, we investigated their role in host acceptance. We quantified these compounds in different willow species and correlated PG content with oviposition preference under lab and natural field conditions. We demonstrated a major role for contact cues in triggering N. oligospilus egg laying on the leaf surface of preferred willow genotypes. Firstly cues are sensed by antennae, determining to leave or stay on the leaf. After that, sensing is performed by abdominal cerci, which finally triggers egg laying. The lack of PGs in non-preferred species and the significant correlation observed between PGs, natural damage and oviposition preference suggest a role for these compounds in host selection. Our study suggests that in specialist feeders, secondary compounds normally acting as defenses can actually act as a susceptibility factor by triggering specific insect behavior for oviposition. These defensive compounds could be selected against to increase resistance.

Glyphosate affects the larval development of honey bees depending on the susceptibility of colonies.

As the main agricultural insect pollinator, the honey bee (Apis mellifera) is exposed to a number of agrochemicals, including glyphosate (GLY), the most widely used herbicide. Actually, GLY has been detected in honey and bee pollen baskets. However, its impact on the honey bee brood is poorly explored. Therefore, we assessed the effects of GLY on larval development under chronic exposure during in vitro rearing. Even though this procedure does not account for social compensatory mechanisms such as brood care by adult workers, it allows us to control the herbicide dose, homogenize nutrition and minimize environmental stress. Our results show that brood fed with food containing GLY traces (1.25-5.0 mg per litre of food) had a higher proportion of larvae with delayed moulting and reduced weight. Our assessment also indicates a non-monotonic dose-response and variability in the effects among colonies. Differences in genetic diversity could explain the variation in susceptibility to GLY. Accordingly, the transcription of immune/detoxifying genes in the guts of larvae exposed to GLY was variably regulated among the colonies studied. Consequently, under laboratory conditions, the response of honey bees to GLY indicates that it is a stressor that affects larval development depending on individual and colony susceptibility.

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.

An Early Sensitive Period Induces Long-Lasting Plasticity in the Honeybee Nervous System.

The effect of early experiences on the brain during a sensitive period exerts a long-lasting influence on the mature individual. Despite behavioral and neural plasticity caused by early experiences having been reported in the honeybee Apis mellifera, the presence of a sensitive period in which associative experiences lead to pronounced modifications in the adult nervous system is still unclear. Laboratory-reared bees were fed with scented food within specific temporal windows and were assessed for memory retention, in the regulation of gene expression related to the synaptic formation and in the olfactory perception of their antennae at 17 days of age. Bees were able to retain a food-odor association acquired 5-8 days after emergence, but not before, and showed better retention than those exposed to an odor at 9-12 days. In the brain, the odor-rewarded experiences that occurred at 5-8 days of age boosted the expression levels of the cell adhesion proteins neurexin 1 (Nrx1) and neuroligin 2 (Nlg2) involved in synaptic strength. At the antennae, the experiences increased the electrical response to a novel odor but not to the one experienced. Therefore, a sensitive period that induces long-lasting behavioral, functional and structural changes is found in adult honeybees.

Solar UV-B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field-grown soybean.

Solar UV-B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV-B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense-related responses in undamaged and Anticarsia gemmatalis larvae-damaged leaves of two soybean cultivars grown under attenuated or full solar UV-B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV-B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane-carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field-grown soybean isoflavonoids were regulated by both herbivory and solar UV-B inducible ET, whereas flavonols were regulated by solar UV-B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV-B-mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.

Solar UV-B radiation modulates chemical defenses against Anticarsia gemmatalis larvae in leaves of field-grown soybean.

Although it is well known that solar ultraviolet B (UV-B) radiation enhances plant defenses, there is less knowledge about traits that define insect resistance in field-grown soybean. Here we study the effects of solar UV-B radiation on: a) the induction of phenolic compounds and trypsin proteinase inhibitors (TPI) in soybean undamaged leaves or damaged by Anticarsia gemmatalis neonates during six days, and b) the survival and mass gain of A. gemmatalis larvae that fed on soybean foliage. Two soybean cultivars (cv.), Charata and Williams, were grown under plastic with different transmittance to solar UV-B radiation, which generated two treatments: ambient UV-B (UVB+) and reduced UV-B (UVB-) radiation. Solar UV-B radiation decreased survivorship by 30% and mass gain by 45% of larvae that fed on cv. Charata, but no effect was found in those larvae that fed on cv. Williams. TPI activity and malonyl genistin were induced by A. gemmatalis damage in both cultivars, but solar UV-B radiation and damage only synergistically increased the induction of these compounds in cv. Williams. Although TPI activity and genistein derivatives were induced by herbivory, these results did not explain the differences found in survivorship and mass gain of larvae that fed on cv. Charata. However, we found a positive association between lower larval performance and the presence of two quercetin triglycosides and a kaempferol triglycoside in foliage of cv. Charata, which were identified by HPLC-DAD/MS2. We conclude that exclusion of solar UV-B radiation reduce resistance to A. gemmatalis, due to a reduction in flavonol concentration in a cultivar that has low levels of genistein derivatives like cv. Charata.

Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12-oxo-phytodienoic acid, mitogen-activated protein kinase 4 and cytokinin perception.

Herbivory-induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore-specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (AC ) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1-5 ribulose-1,5-bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion-elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12-oxo-phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen-activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen-activated protein kinase 4 in CO2 -mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid-mediated defence responses, also elicit 12-oxo-phytodienoic acid-mediated changes in stomatal conductance and AC , an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

BACKGROUND: Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. RESULTS: This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. CONCLUSION: This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean. © 2015 Society of Chemical Industry.

Both Volatiles and Cuticular Plant Compounds Determine Oviposition of the Willow Sawfly Nematus oligospilus on Leaves of Salix spp. (Salicaceae).

Plant volatile organic compounds play a role in selection of host plants by herbivorous insects. Once the insect reaches the plant, contact cues determine host acceptance. Although the willow sawfly Nematus oligospilus (Hymenoptera: Tenthredinidae) can differentiate among willow genotypes, no knowledge is available on the cues used by this insect to seek and accept the host plant. In this study, we recorded behavioral orientation in a Y-tube olfactometer of willow sawfly females to volatiles of the highly preferred genotype Salix nigra and the non-preferred genotype S. viminalis. The volatiles released by undamaged willows of each genotype were analyzed by coupled gas chromatography-mass spectrometry. Contact cues were evaluated first by oviposition preference bioassays after selective leaf wax removal, and then by studying the micromorphology of abaxial and adaxial leaf surfaces and their chemical composition. Willow sawfly females oriented preferentially to S. nigra volatiles, which contained more than 3 times the amount of volatiles than that collected from S. viminalis. Analysis of volatiles showed significant differences in amounts of (Z) and (E)-ß-ocimene, undecane, decanal, and ß-caryophyllene. The adaxial leaf surface of S. nigra was less preferred after wax removal, suggesting a role of cuticular waxes for oviposition acceptance. No differences were found among the micromorphology of leaf surfaces between preferred and non-preferred genotypes. The chemical analysis of cuticular waxes showed that the abaxial leaf surface of S. viminalis, which is completely avoided for oviposition, possessed 97% of alkanes. The accepted leaf surfaces contained a more diverse wax profile including alcohols, acids, and esters. Thus, non-alkane wax compounds might be related to oviposition. In sum, our study suggests that several cues act in concert to provide oviposition cues for the sawfly N. oligospilus: females are attracted to volatiles from a distance, and once alighting on the plant, they seek specific chemical contact cues in order to lay eggs.