The proteomic landscape of fall armyworm oral secretion reveals its role in plant adaptation.

BACKGROUND: The fall armyworm (FAW, Spodoptera frugiperda (J.E. Smith)) is a polyphagous agricultural pest with rapidly evolving adaptations to host plants. We found the oral secretion (OS) of FAW from different plants influences plant defense response differentially, suggesting its role in adapting to host plants. However, the protein expression profile of FAW OS respond to different plants is largely unknown. RESULTS: Here, from the mass spectrometry assay, we identified a total of 256 proteins in the OS of FAW fed on cotton (Gossypium hirsutum L.), tobacco (Nicotiana benthamiana Domin), maize (Zea mays L.) and artificial diet. The FAW OS primarily comprise of 60 proteases, 32 esterases and 92 non-enzymatic proteins. It displays high plasticity across different diets. We found that more than half of the esterases are lipases which have been reported as insect elicitors to enhance plant defense response. The lipase accumulation in cotton-fed larvae was the highest, followed by maize-fed larvae. In the presence of lipase inhibitors, the enhanced induction on defense genes in wounded leaves by OS was attenuated. However, the putative effectors were most highly accumulated in the OS from FAW larvae fed on maize compared to those fed on other diets. We identified that one of them (VRLP4) reduces the OS-mediated induction on defense genes in wounded leaves. CONCLUSION: Together, our investigation presents the proteomic landscape of the OS of FAW influenced by different diets and reveals diet-mediated plasticity of OS is involved in FAW adaptation to host plants. © 2024 Society of Chemical Industry.

From weeds to natural enemies: implications of weed cultivation and biopesticides for organic onion production.

Weed management is challenging for vegetable crops that are highly sensitive to weed competition, such as onions. Thrips (Thysanoptera: Thripidae) are major insect pests of onions, causing damage through feeding, and vectoring bacterial pathogens causing bulb rot. Both thrips and their associated pathogens are known to survive on many weed species in onion growing regions. Combining weeding with biopesticides may synergistically manage thrips and reduce disease prevalence. However, disturbances from weeding may negatively impact natural enemies. We estimated the effects of organic weed management and biopesticides on weed density, thrips and natural enemy activity, disease severity, and yield. The experiment was a randomized complete block design, with 4 replications of each weeding (control, tine-weeded twice, tine-weeded 4 times, and hand-weeded) and biopesticide (control, OxiDate 2.0, Serenade) combination. Arthropods were monitored using yellow sticky cards, and weed counts, marketable yield, and bulb rot prevalence were estimated. Hand-weeding resulted in the lowest weed density and thrips abundance. Additionally, hand-weeding produced a 9× higher yield compared to all other treatments. Significant interactions were observed between tine-weeding and biopesticide treatments on the prevalence of bulb rot. Natural enemy abundance was slightly negatively impacted by weeding, dependent on the year. DNA metabarcoding results showed high parasitoid diversity in this onion system and high numbers of reads for multiple genera containing important known biological control agents. Our study suggests hand-weeding is necessary in the southeast for maximum onion yield. Future research should focus on exploring the impact of management on natural enemy communities in onion systems on a large scale.

A semiochemical view of the ecology of the seed beetle Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae, Bruchinae).

The dried bean beetle, Acanthoscelides obtectus, is an economically important pest of stored legumes worldwide. Tracking the human-aided dispersion of its primary hosts, the Phaseolus vulgaris beans, it is now widespread in most bean-growing areas of the tropics and subtropics. In temperate regions where it can only occasionally overwinter in the field, A. obtectus proliferates in granaries, having multiple generations a year. Despite its negative impact on food production, no sensitive detection or monitoring tools exist, and the reduction of local populations still relies primarily on inorganic insecticides as fumigating agents. However, in the quest to produce more nutritious food more sustainably and healthily, the development of environmentally benign crop protection methods is vital against A. obtectus. For this, knowledge of the biology and chemistry of both the host plant and its herbivore will underpin the development of, among others, chemical ecology-based approaches to form an essential part of the toolkit of integrated bruchid management. We review the semiochemistry of the mate- and host-finding behaviour of A. obtectus and provide new information about the effect of seed chemistry on the sensory and behavioural ecology of host acceptance and larval development.

Implications of cell wall immunocytochemical profiles on the structural and functional traits of root and stem galls induced by Eriosoma lanigerum on Malus domestica.

Alterations in cell wall composition imply in new structural and functional traits in gall developmental sites, even when the inducer is a sucking exophytophagous insect with strict feeding sites as the aphid associated to Malus domestica Borkh. This host plant is an economically important, fruit-bearing species, susceptible to gall induction by the sucking aphid Eriosoma lanigerum Hausmann, 1802. Herein, the immunocytochemical detection of arabinogalactan-proteins (AGPs), pectins, and hemicelluloses using monoclonal antibodies was performed in samples of non-galled roots and stems, and of root and stem galls on M. domestica. The dynamics of these cell wall components was discussed under the structural and functional traits of the galls proximal, median, and distal regions, according to the proximity of E. lanigerum colony feeding site. In the proximal region, the epitopes of AGPs and homogalacturonans (HGs) are related to cell growth and divisions, which result in the overproduction of parenchyma cells both in root and stem galls. In the proximal and median regions, the co-occurrence of HGs and arabinans in the cell walls of parenchyma and secondary tissues favors the nutrient flow and water-holding capacity, while the xylogalacturonans and hemicelluloses may function as additional carbohydrate resources to E. lanigerum. The immunocytochemical profile of the cell walls support the feeding activity of E. lanigerum mainly in the gall proximal region. The similarity of the cell wall components of the gall distal region and the non-galled portions, both in roots and stems, relates to the decrease of the cecidogenetic field the more distant the E. lanigerum colony is.

Rich specialized insect damage on Pliocene leaves from the Mahuadanr Valley (India) growing under a warm climate with weak seasonality.

Plant-insect interactions play a crucial role in shaping terrestrial ecosystems, influencing abundance and distribution of plant species. In the present study, we investigated leaf-mining patterns on fossil leaves from Pliocene strata of the Mahuadanr Valley, Jharkhand, eastern India, deposited under a seasonal tropical climate, and reported complex interactions between plants and insects. We identified 11 distinct mining morphotypes. These morphotypes were mainly found on Dipterocarpaceae, Fabaceae, Lauraceae, and Moraceae; similar mining traces were also observed in the contemporary vegetation surrounding the fossil site. Although mining richness was relatively high, only 2.6% of all leaves in the fossil assemblage were mined. We compared mining richness and abundance values with previously reported values for galling. While richness was slightly lower for galling, almost 50% of all fossil leaves were galled. A literature survey on mining and galling patterns in modern vegetation suggests that there is no global explanation for richness of mining or gall-inducing insects. Thus, low nutrient availability in the ancient forest, dominance of semideciduous leaves with hard texture, and different habitats in the same forest ecosystem, such as well-drained forests and riparian stands, may all have favored different types of specialized plant-insect interactions.

GC-MS-based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect.

Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.

Construction of Host Plant Insect-Resistance Mutant Library by High-Throughput CRISPR/Cas9 System and Identification of A Broad-Spectrum Insect Resistance Gene.

Insects pose significant challenges in cotton-producing regions. Here, they describe a high-throughput CRISPR/Cas9-mediated large-scale mutagenesis library targeting endogenous insect-resistance-related genes in cotton. This library targeted 502 previously identified genes using 968 sgRNAs, generated ≈2000 T0 plants and achieved 97.29% genome editing with efficient heredity, reaching upto 84.78%. Several potential resistance-related mutants (10% of 200 lines) their identified that may contribute to cotton-insect molecular interaction. Among these, they selected 139 and 144 lines showing decreased resistance to pest infestation and targeting major latex-like protein 423 (GhMLP423) for in-depth study. Overexpression of GhMLP423 enhanced insect resistance by activating the plant systemic acquired resistance (SAR) of salicylic acid (SA) and pathogenesis-related (PR) genes. This activation is induced by an elevation of cytosolic calcium [Ca2+ ]cyt flux eliciting reactive oxygen species (ROS), which their demoted in GhMLP423 knockout (CR) plants. Protein-protein interaction assays revealed that GhMLP423 interacted with a human epidermal growth factor receptor substrate15 (EPS15) protein at the cell membrane. Together, they regulated the systemically propagating waves of Ca2+ and ROS, which in turn induced SAR. Collectively, this large-scale mutagenesis library provides an efficient strategy for functional genomics research of polyploid plant species and serves as a solid platform for genetic engineering of insect resistance.

Transcriptomic and metabolomic analyses reveals keys genes and metabolic pathways in tea (Camellia sinensis) against six-spotted spider mite (Eotetranychus Sexmaculatus).

BACKGROUND: Six-spotted spider mite (Eotetranychus sexmaculatus) is one of the most damaging pests of tea (Camellia sinensis). E. sexmaculatus causes great economic loss and affects tea quality adversely. In response to pests, such as spider mites, tea plants have evolved resistance mechanisms, such as expression of defense-related genes and defense-related metabolites. RESULTS: To evaluate the biochemical and molecular mechanisms of resistance in C. sinensis against spider mites, "Tianfu-5" (resistant to E. sexmaculatus) and "Fuding Dabai" (susceptible to E. sexmaculatus) were inoculated with spider mites. Transcriptomics and metabolomics based on RNA-Seq and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technology were used to analyze changes in gene expression and metabolite content, respectively. RNA-Seq data analysis revealed that 246 to 3,986 differentially expressed genes (DEGs) were identified in multiple compared groups, and these DEGs were significantly enriched in various pathways, such as phenylpropanoid and flavonoid biosynthesis, plant-pathogen interactions, MAPK signaling, and plant hormone signaling. Additionally, the metabolome data detected 2,220 metabolites, with 194 to 260 differentially abundant metabolites (DAMs) identified in multiple compared groups, including phenylalanine, lignin, salicylic acid, and jasmonic acid. The combined analysis of RNA-Seq and metabolomic data indicated that phenylpropanoid and flavonoid biosynthesis, MAPK signaling, and Ca2+-mediated PR-1 signaling pathways may contribute to spider mite resistance. CONCLUSIONS: Our findings provide insights for identifying insect-induced genes and metabolites and form a basis for studies on mechanisms of host defense against spider mites in C. sinensis. The candidate genes and metabolites identified will be a valuable resource for tea breeding in response to biotic stress.

Mannas, unique products of a dynamic insect-plant interaction: Biodiversity, conservation and ethnopharmacological considerations.

Biodiversity keeping increases the flexibility, interaction and adaptation of different ecosystems with the environment which benefits all organisms, including humans. This diversity can be maintained through different paths including co-evolution between insects and plants. One of these unique interactions leads to the production of "Mannas" in Iran, which have biological and ethno-medicinal importance. Considering the crises of biodiversity and the rapid extinction of species, in this research, we aimed to review the latest scientific findings about mannas and their biological, medicinal and bio perspective considerations. Until August 2023, all English publications in Web of Science, Science Direct, PubMed, Google Scholar and Scopus, as well as Persian databases such as Magiran, Iranmedex, Medlib, SID were surveyed using various search terms such as manna, angabin, sweet secretions and plant-insect interference. Articles that studied manna production from plants or provided a comprehensive description of host plants and manna producing insects were included in the study. In total, out of more than 180 reviewed articles, 113 articles met the inclusion criteria and 7 known mannas (Bidkhesht, Gaz-e-Alafi, Gaz-e-Khansar, Gaz-e-Shahdad, Shirkhesht, Shekartigal, and Taranjebin) have been explained here. This review deals with different aspects of special interactions between insects and plants that lead to the production of manna and presents different uses of manna from medicinal, ethnobotanical, health, conservation and bio perspective points of view. In addition, the changes in the population of manna-breeding insects and host plants are taken into consideration and influencing factors including loss of their growth conditions and climatic conditions, animal breeding in the region, inappropriate exploitation methods and host preference phenomenon which increase the risk of destruction of these natural products have been given. These mannas cannot be produced in any way, and maintaining their production conditions requires environmental care and providing necessary training.

Identification of Potential Gene Targets for Suppressing Oviposition in Holotrichia parallela Using Comparative Transcriptome Analysis.

Holotrichia parallela is an important plant pest. Comparative feeding experiments showed that the egg production, oviposition duration and survival rate of H. parallela beetles were significantly higher when they fed on elm leaves than when they fed on willow or purpus privet leaves. RNA sequencing was used to determine transcriptomic changes associated with oviposition. Comparative transcriptome analysis revealed that the beetles that fed on elm and willow had a total of 171 genes with differential expression. When the beetles fed on elm and purpus privet, 3568 genes had differential expression. The vitellogenesis, ovarian serine protease, odorant-binding proteins, acyl-CoA synthetase and follicle cell proteins were commonly upregulated genes in elm-fed beetles compared with those fed on willow/purpus privet leaves. The involvement of the follicle cell protein 3C gene in the regulation of oviposition was confirmed using RNA interference. The results provide insights into the molecular mechanisms underlying oviposition in H. parallela feeding on different host plants. This study also describes a method for identifying potentially effective genes for pest control.

'Candidatus Phytoplasma ziziphi' Changes the Metabolite Composition of Jujube Tree Leaves and Affects the Feeding Behavior of Its Insect Vector Hishimonus hamatus Kuoh.

Hishimonus hamatus Kuoh is a leafhopper species native to China that feeds on Chinese jujube leaves. This leafhopper species has been verified to transmit jujube witches' broom (JWB) disease, caused by phytoplasma, a fatal plant pathogen, which belongs to the phytoplasma subgroup 16SrV-B. The transmission of JWB phytoplasma largely relies on the feeding behavior of piercing-sucking leafhoppers. However, the specific mechanisms behind how and why the infection of JWB influences the feeding behavior of these leafhoppers are not fully understood. To address this, a study was conducted to compare the feeding patterns of H. hamatus when feeding JWB-infested jujube leaves to healthy leaves using the electrical penetration graph (EPG) technique. Then, a widely targeted metabolome analysis was performed to identify differences in the metabolite composition of JWB-infected jujube leaves and that of healthy jujube leaves. The results of EPG analyses revealed that when feeding on JWB-infected jujube leaves, H. hamatus exhibited an increased frequency of phloem ingestion and spent longer in the phloem feeding phase compared to when feeding on healthy leaves. In addition, the results of metabolomic analyses showed that JWB-infected leaves accumulated higher levels of small-molecular carbohydrates, free amino acids, and free fatty acids, as well as lower levels of lignans, coumarins and triterpenoids compared to healthy leaves. The above results indicated that the H. hamatus preferentially fed on the phloem of infected leaves, which seems to be linked to the transmission of the JWB phytoplasma. The results of metabolomic analyses partially imply that the chemical compounds might play a role in making the infected leaves more attractive to H. hamatus for feeding.

Exploring spatiotemporal dynamics of flower visitor association pattern on two Avicennia mangroves: a network approach.

Plant-flower visitor interaction is one of the most important relationships regarding the co-existence of the floral and faunal communities. The implication of network approaches is an efficient way to understand the impact of community structure on ecosystem functionality. To understand the association pattern of flower visitors, we performed this study on Avicennia officinalis and Avicennia marina mangroves from the islands of Indian Sundarban over three consecutive years. We found that visiting time and sites (islands) influenced the abundance of visitors. The bipartite networks showed a significant generalized structure for both site-visitor and visiting time-visitor networks where the strength and specialization of visitor species showed a highly and moderately significant positive correlation between both networks respectively. All the site-wise visiting time-visitor networks and year-wise site-visitor networks were significantly modular in structure. For both the plants, most of the visitors showed a generalized association pattern among islands and also among visiting times. Additionally, the study of the foraging behavior of dominant visitors showed Apis dorsata and Apis mellifera as the potential visitors for these plants. Our results showed that flower visitor networks are spatiotemporally dynamic. The interactions of visitors with flowers at different times influence their contribution to the network for becoming a generalist or peripheral species in the context of their visiting time, which may subsequently change over islands. This approach will help to devise more precise plant species-specific conservation strategies by understanding the contribution of visitors through the spatiotemporal context.

Plant water limitation and its impact on the oviposition preferences of the monarch butterfly (Lepidoptera: Nymphalidae).

Intensifying drought conditions across the western United States due to global climate change are altering plant-insect interactions. Specialist herbivores must find their host plants within a matrix of nonhosts, and thus often rely upon specific plant secondary chemistry for host location and oviposition cues. Climate-induced alterations to plant chemistry could thus affect female selection of larval food plants. Here, we investigated whether host-plant water limitation influenced oviposition preference in a threatened invertebrate: the monarch butterfly (Danaus plexippus). We found that females deposited more eggs on reduced-water than on well-watered narrowleaf milkweed plants (Asclepias fascicularis), but we could not attribute this change to any specific change in plant chemistry. Specialist herbivores, such as the monarch butterfly, which are tightly linked to specific plant cues, may experience shift in preferences under global-change conditions. Understanding oviposition preferences will be important to directing ongoing habitat restoration activities for this declining insect.

Genome-wide association study of aphid abundance highlights a locus affecting plant growth and flowering in Arabidopsis thaliana.

Plant life-history traits, such as size and flowering, contribute to shaping variation in herbivore abundance. Although plant genes involved in physical and chemical traits have been well studied, less is known about the loci linking plant life-history traits and herbivore abundance. Here, we conducted a genome-wide association study (GWAS) of aphid abundance in a field population of Arabidopsis thaliana. This GWAS of aphid abundance detected a relatively rare but significant variant on the third chromosome of A. thaliana, which was also suggestively but non-significantly associated with the presence or absence of inflorescence. Out of candidate genes near this significant variant, a mutant of a ribosomal gene (AT3G13882) exhibited slower growth and later flowering than a wild type under laboratory conditions. A no-choice assay with the turnip aphid, Lipaphis erysimi, found that aphids were unable to successfully establish on the mutant. Our GWAS of aphid abundance unexpectedly found a locus affecting plant growth and flowering.

Transcriptome features of stone cell development in weevil-resistant and susceptible Sitka spruce.

Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation.

Integrated transcriptomics and metabolomics analysis provide insight into the resistance response of rice against brown planthopper.

Introduction: The brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most economically significant pests of rice. The Bph30 gene has been successfully cloned and conferred rice with broad-spectrum resistance to BPH. However, the molecular mechanisms by which Bph30 enhances resistance to BPH remain poorly understood. Methods: Here, we conducted a transcriptomic and metabolomic analysis of Bph30-transgenic (BPH30T) and BPH-susceptible Nipponbare plants to elucidate the response of Bph30 to BPH infestation. Results: Transcriptomic analyses revealed that the pathway of plant hormone signal transduction enriched exclusively in Nipponbare, and the greatest number of differentially expressed genes (DEGs) were involved in indole 3-acetic acid (IAA) signal transduction. Analysis of differentially accumulated metabolites (DAMs) revealed that DAMs involved in the amino acids and derivatives category were down-regulated in BPH30T plants following BPH feeding, and the great majority of DAMs in flavonoids category displayed the trend of increasing in BPH30T plants; the opposite pattern was observed in Nipponbare plants. Combined transcriptomics and metabolomics analysis revealed that the pathways of amino acids biosynthesis, plant hormone signal transduction, phenylpropanoid biosynthesis and flavonoid biosynthesis were enriched. The content of IAA significantly decreased in BPH30T plants following BPH feeding, and the content of IAA remained unchanged in Nipponbare. The exogenous application of IAA weakened the BPH resistance conferred by Bph30. Discussion: Our results indicated that Bph30 might coordinate the movement of primary and secondary metabolites and hormones in plants via the shikimate pathway to enhance the resistance of rice to BPH. Our results have important reference significance for the resistance mechanisms analysis and the efficient utilization of major BPH-resistance genes.

Structural patterns of Lepidoptera galls and the case of Andescecidium parrai (Cecidosidae) galls on Schinus polygama (Anacardiaceae).

Gall anatomical and metabolic peculiarities are determined by the feeding habit of the gall inducer, but develop under the constraints of the host plants. The chewing habit of the Lepidoptera larvae imposes a high impact on the host plant cells, and supposedly drives peculiar structural and histochemical patterns. So, our starting point was the search of such patterns in literature, and the test of these traits on the Andescecidium parrai (Cecidosidae)-Schinus polygama (Anacardiaceae) system, as a case study in Chilean flora. The literature on the structure of lepidopteran galls in the temperate and tropical regions comprises 13 works, describing stems as the most frequent host organs, followed by leaves, buds, and flowers. As common structural traits of Lepidoptera galls, the literature converge in describing the processes of cell hypertrophy and hyperplasia, resulting in a variable number of common storage parenchyma layers, interspersed by the redifferentiated sclerenchyma, vascular, and typical nutritive cells around the larval chamber. These nutritive cells accumulate lipids and proteins, which support the lepidopteran larvae nutrition. As expected, the A. parrai galls follow the patterns herein described for the lepidoptera-induced galls, but with peculiarities associated with its host organ. Even though the Lepidoptera galls have destructive mouthparts and can induce large and complex galls, they cannot alter important conservative features of their hosts' organs.

Macroecological diversification of ants is linked to angiosperm evolution.

Ants are abundant, diverse, and occupy nearly all habitats and regions of the world. Previous work has demonstrated that ant diversification coincided with the rise of the angiosperms, and that several plant traits evolved as ants began to expand their nesting and foraging habits. In this study, we investigate whether associations with plants enabled niche expansion and are linked to climatic niche evolution in ants. Our analysis of over 1,400 ant species reveals that ancestral expansion from forest floors into the canopy and out into non-forested habitats closely followed evolutionary innovations in angiosperms. Several Paleogene-Neogene ant lineages independently diversified in non-forested habitats on multiple continents, tracking the evolution and expansion of elaiosome-bearing and arid-adapted angiosperms. The evolution of arboreal nesting tracked shifts in angiosperm physiology associated with the onset of everwet tropical rainforests, and climatic optima and rates of climatic niche evolution were linked to nesting location, with arboreally nesting groups having warmer and less seasonal climatic optima, and lower rates of climatic niche evolution. Our work further underscores the varied paths by which niche diversification occurred in ants, and how angiosperms influenced the ecological and evolutionary trajectories of interacting lineages.

An integrated leaf trait analysis of two Paleogene leaf floras.

Objectives: This study presents the Integrated Leaf Trait Analysis (ILTA), a workflow for the combined application of methodologies in leaf trait and insect herbivory analyses on fossil dicot leaf assemblages. The objectives were (1) to record the leaf morphological variability, (2) to describe the herbivory pattern on fossil leaves, (3) to explore relations between leaf morphological trait combination types (TCTs), quantitative leaf traits, and other plant characteristics (e.g., phenology), and (4) to explore relations of leaf traits and insect herbivory. Material and Methods: The leaves of the early Oligocene floras Seifhennersdorf (Saxony, Germany) and Suletice-Berand (Ústí nad Labem Region, Czech Republic) were analyzed. The TCT approach was used to record the leaf morphological patterns. Metrics based on damage types on leaves were used to describe the kind and extent of insect herbivory. The leaf assemblages were characterized quantitatively (e.g., leaf area and leaf mass per area (LMA)) based on subsamples of 400 leaves per site. Multivariate analyses were performed to explore trait variations. Results: In Seifhennersdorf, toothed leaves of TCT F from deciduous fossil-species are most frequent. The flora of Suletice-Berand is dominated by evergreen fossil-species, which is reflected by the occurrence of toothed and untoothed leaves with closed secondary venation types (TCTs A or E). Significant differences are observed for mean leaf area and LMA, with larger leaves tending to lower LMA in Seifhennersdorf and smaller leaves tending to higher LMA in Suletice-Berand. The frequency and richness of damage types are significantly higher in Suletice-Berand than in Seifhennersdorf. In Seifhennersdorf, the evidence of damage types is highest on deciduous fossil-species, whereas it is highest on evergreen fossil-species in Suletice-Berand. Overall, insect herbivory tends to be more frequently to occur on toothed leaves (TCTs E, F, and P) that are of low LMA. The frequency, richness, and occurrence of damage types vary among fossil-species with similar phenology and TCT. In general, they are highest on leaves of abundant fossil-species. Discussion: TCTs reflect the diversity and abundance of leaf architectural types of fossil floras. Differences in TCT proportions and quantitative leaf traits may be consistent with local variations in the proportion of broad-leaved deciduous and evergreen elements in the ecotonal vegetation of the early Oligocene. A correlation between leaf size, LMA, and fossil-species indicates that trait variations are partly dependent on the taxonomic composition. Leaf morphology or TCTs itself cannot explain the difference in insect herbivory on leaves. It is a more complex relationship where leaf morphology, LMA, phenology, and taxonomic affiliation are crucial.

'Sharpshooter' in Botanic Garden: the tale of a rare plant-insect interaction.

Here we report a unique plant-insect interaction between the leafhopper Aloka depressa (tribe Phlogisini) and the host liana, Diploclisia glaucescens, from a Botanic Garden located at the southern edge of Western Ghats in India. Field observations and SEM micrographs were employed to derive evidences on this rare plant-insect interaction. 20-Hydroxyecdysone (20E), insect moulting hormone, was detected and quantified in the host plant D. glaucescens using HPTLC-densitometry. 20E was isolated and characterized from D. glaucescens using column chromatography, 1H-, 13C-NMR and HR-MS. 20E was also detected in A. depressa excrement using HPTLC-densitometry. The leafhopper A. depressa is functioning as a 'sharpshooter' drawing nutrients from the host liana, D. glaucescens, and flinging the waste fluid as droplets through their tail ends. SEM micrographs of A. depressa revealed its external morphological features, characteristic of a sharpshooter. We quantified 20E (0.44-1.44%, dry wt.) in various parts of D. glaucescens. 20E (1.47%, dry wt.) was also detected in the excrement of A. depressa. This plant (D. glaucescens)-insect (A. depressa) association crucially is not damaging the host liana. Considering the diseases caused by sharpshooting leafhoppers in the Americas, this association and the survival of the host plant (D. glaucescens) is illustrating a unique plant-insect interaction.