Comparative transcriptome reprogramming in oak galls containing asexual or sexual generations of gall wasps.

Oak gall wasps have evolved strategies to manipulate the developmental pathways of their host to induce gall formation. This provides shelter and nutrients for the developing larva. Galls are entirely host tissue; however, the initiation, development, and physical appearance are controlled by the inducer. The underlying molecular mechanisms of gall formation, by which one or a small number of cells are reprogrammed and commit to a novel developmental path, are poorly understood. In this study, we sought a deeper insight into the molecular underpinnings of this process. Oak gall wasps have two generations each year, one sexual, and one asexual. Galls formed by these two generations exhibit a markedly different appearance. We sequenced transcriptomes of both the asexual and sexual generations of Neuroterus quercusbaccarum and Neuroterus numismalis. We then deployed Nanopore sequencing to generate long-read sequences to test the hypothesis that gall wasps introduce DNA insertions to determine gall development. We detected potential genome rearrangements but did not uncover any non-host DNA insertions. Transcriptome analysis revealed that transcriptomes of the sexual generations of distinct species of wasp are more similar than inter-generational comparisons from the same species of wasp. Our results highlight the intricate interplay between the host leaves and gall development, suggesting that season and requirements of the gall structure play a larger role than species in controlling gall development and structure.

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

From Inquilines to Gall Inducers: Genomic Signature of a Life-Style Transition in Synergus Gall Wasps.

Gall wasps (Hymenoptera: Cynipidae) induce complex galls on oaks, roses, and other plants, but the mechanism of gall induction is still unknown. Here, we take a comparative genomic approach to revealing the genetic basis of gall induction. We focus on Synergus itoensis, a species that induces galls inside oak acorns. Previous studies suggested that this species evolved the ability to initiate gall formation recently, as it is deeply nested within the genus Synergus, whose members are mostly inquilines that develop inside the galls of other species. We compared the genome of S. itoensis with that of three related Synergus inquilines to identify genomic changes associated with the origin of gall induction. We used a novel Bayesian selection analysis, which accounts for branch-specific and gene-specific selection effects, to search for signatures of selection in 7,600 single-copy orthologous genes shared by the four Synergus species. We found that the terminal branch leading to S. itoensis had more genes with a significantly elevated dN/dS ratio (positive signature genes) than the other terminal branches in the tree; the S. itoensis branch also had more genes with a significantly decreased dN/dS ratio. Gene set enrichment analysis showed that the positive signature gene set of S. itoensis, unlike those of the inquiline species, is enriched in several biological process Gene Ontology terms, the most prominent of which is "Ovarian Follicle Cell Development." Our results indicate that the origin of gall induction is associated with distinct genomic changes, and provide a good starting point for further characterization of the genes involved.

Changes in oak (Quercus robur) photosynthesis after winter moth (Operophtera brumata) herbivory are not explained by changes in chemical or structural leaf traits.

Insect herbivores have the potential to change both physical and chemical traits of their host plant. Although the impacts of herbivores on their hosts have been widely studied, experiments assessing changes in multiple leaf traits or functions simultaneously are still rare. We experimentally tested whether herbivory by winter moth (Operophtera brumata) caterpillars and mechanical leaf wounding changed leaf mass per area, leaf area, leaf carbon and nitrogen content, and the concentrations of 27 polyphenol compounds on oak (Quercus robur) leaves. To investigate how potential changes in the studied traits affect leaf functioning, we related the traits to the rates of leaf photosynthesis and respiration. Overall, we did not detect any clear effects of herbivory or mechanical leaf damage on the chemical or physical leaf traits, despite clear effect of herbivory on photosynthesis. Rather, the trait variation was primarily driven by variation between individual trees. Only leaf nitrogen content and a subset of the studied polyphenol compounds correlated with photosynthesis and leaf respiration. Our results suggest that in our study system, abiotic conditions related to the growth location, variation between tree individuals, and seasonal trends in plant physiology are more important than herbivory in determining the distribution and composition of leaf chemical and structural traits.

Effect of Neuroterus quercusbaccarum (L.) galls on physiological and biochemical response of Quercus robur leaves.

Gall formation is associated with multiple changes in plant cells, which still requires a better understanding. In this study, galls caused by sexual generation (♀♂) of Neuroterus quercusbaccarum (L.) (Hymenoptera: Cynipidae) on pedunculate oak trees (Quercus robur L.) were used as a model. Cytoplasmic membrane condition, concentration of hydrogen peroxide (H2O2), the activity of antioxidant enzymes and amino acid decarboxylase as well as chlorophyll fluorescence parameters were determined. Changes in physiological and biochemical parameters were analyzed in foliar tissues with galls and gall tissues themselves and compared to control. The presence of galls on oak leaves caused an increase of lipid peroxidation level. A significant decline in H2O2 and TBARS content with the reduction of guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) activity were observed in gall tissues. The activity amino acid decarboxylase, i.e., LDC, ODC and TyDC varied between samples, which may affect the content of amino acids. The presence of N. quercusbaccarum galls caused an insignificant increase of the chlorophylls, carotenoids and anthocyanin contents, while the content of pigments and their ratios in gall tissues was extremely low. Moreover, photosynthetic parameters (F0, Fm, Fv/Fm, Y, qP) were significantly decreased. Data generated in this study indicate that the development of N. quercusbaccarum galls on pedunculate oak leaves has a negative effect on host plant related to the disruption of cell membrane integrity, disturbance of photosynthesis and reduction of the antioxidant potential of the host plant.

Home economics in an oak gall: behavioural and chemical immune strategies against a fungal pathogen in Temnothorax ant nests.

Nest architecture is a fundamental character shaping immune strategies of social insects. The arboreal ant Temnothorax unifasciatus nests in cavities such as oak galls where the entire colony lives in a unique small chamber. In these conditions, physiological and behavioural strategies likely prevail over compartmentalisation and are presumably tuned with colony size. We designed two experiments to study chemical and behavioural immune strategies against the entomopathogenic fungus Metarhizium anisopliae in colonies of different sizes. First, we compared spore germination and length of germinal tubes inside artificial nests, designed to impede the contact between the ants and the fungus, in colonies of different size. In the absence of direct contact, Temnothorax unifasciatus colonies inhibit fungal growth inside their nests, presumably through volatile compounds. The analysis revealed a positive correlation between fungistatic activity and colony size, indicating that workers of smaller colonies do not invest a higher per capita effort in producing such substances compared to larger colonies. Second, we performed a removal experiment of contaminated and non-contaminated items introduced inside the nests of colonies of different size. Small colonies challenged with contaminated fibres showed an increased removal of all the items (both contaminated and non-contaminated) compared to small colonies challenged with non-contaminated fibres only. Conversely, larger colonies moved items regardless of the presence of the spores inside the nest. Colony size qualitatively affected removal of waste items showing a pathogen elicited reaction in small colonies to optimise the reduced workforce, while the removal behaviour in larger colonies revealed to be expressed constitutively.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Three types of gut bacteria collaborating to improve Kui Jie'an enema treat DSS-induced colitis in mice.

A rising amount of evidences show that gut microbiota is an important factor in mediating the growth of ulcerative colitis (UC), the major product of colon bacteria fermentation butyrate as mediator have effects on the mucosal immune system by expanding regulatory T cells (Treg) in the colon. Turkish galls, an insect gall parasitized on the tree branches of Quercus infectoria Oliv., exhibiting the promising prospect in treating the remedy of UC by regulating the bacteria, whereas its mechanism remains unclear. Here, this work found that three types of gut bacteria collaborating to improve DSS-induced UC in mice after Turkish galls intervention, including putative SCFAs-producing bacteria (PCPB), anti-inflammatory bacteria and harmful bacteria. The Helicobacter, Bilophila, Acinetobacter and Odoribacter, which belong to the harmful bacteria were dramatically increased in UC group, whereas the harmful bacteria were reduced after treatment with Turkish galls. The Allobaculum, Bacteriodes, Blautia, Butyricimonas, belonging to PCPB, were significantly increased after Turkish galls and butyrate intervention, and we also observed that the concentration of butyrate increased with the grows of PCPB. The Bifidobacterium, Lactococcus, which belong to the anti-inflammatory bacteria, were also significantly increased after Turkish galls intervention. Meanwhile, rectal administration of Turkish galls and butyrate could increase mucosa inflammation and diarrhea. The expression of cytokines in the colon was improved by butyrate and Turkish galls treatment group. The percentage of Treg out of CD4+ population was evaluated by flow cytometry after Turkish galls and butyrate intervention. The results suggested that Turkish galls alleviated UC by modulating three types of the gut microbiota, and butyrate may be used to relieve inflammatory. This study may help us to understand the mechanism of Turkish galls in treating UC from the perspective of intestinal flora and also offered a mechanism reference for UC treatment using an insect gall in rich of polyphenolic compounds.

Screening for active constituents in Turkish galls against ulcerative colitis by mass spectrometry guided preparative chromatography strategy: in silico, in vitro and in vivo study.

Turkish galls have been reported to exhibit remedial effects in ulcerative colitis (UC). However, the active constituents of Turkish galls for the treatment of UC remain unclear. The objective of this study was to screen for anti-inflammatory active constituents and clarify their associated molecular mechanisms. Therefore, systems pharmacology was developed to predict the relationship between constituents and the corresponding targets as well as pathways. In addition, mass spectrometry-guided preparative chromatography technique was used for preparing constituents to evaluate the anti-inflammatory activities and the therapeutic efficacy against UC. In silico, active constituents exhibited a remedial effect on UC possibly by regulating multiple pathways and attacking multiple targets, of which those involved mainly in the NF-κB pathway were selected for verification. In vitro, 5 categories of constituents were screened as active constituents by comparing the cytotoxicity and detecting the level of the pro-inflammatory factors of 9 category constituents. In vivo, dextran sulfate sodium (DSS)-induced UC was significantly ameliorated in active constituents-fed mice. The results indicated that the active fraction comprising methyl gallate, digallic acid, di-O-galloyl-ß-d-glucose, and tri-O-galloyl-ß-d-glucose primarily contributed to the treatment of UC. Moreover, active fraction could also inhibit the phosphorylation level of IKKß, thus inhibiting the downstream NF-κB signaling pathway. The approach developed in this study not only clarifies the anti-inflammation effect of Turkish galls but also provides a beneficial reference for the discovery of the base material and functional mechanism of this herbal medicine.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses in A. pernyi.

Biochemical responses induced in galls of three Cynipidae species in oak trees.

Gall-making Cynipidae manipulate the leaves of host plant to form galls where offspring find shelter and food. The relationship between oak gallwasp and biochemical mechanisms of galls still requires a better understanding. So, in this research, protein and phenolic compound contents, as well as the activity of antioxidative enzymes and pathogenesis-related (PR) proteins were determined. Galls caused by asexual generation of Cynips quercusfolii L., Neuroterus numismalis (Fourc.) and N. quercusbaccarum L., as a model were used. All cynipid species modified the protein levels of gall tissues, but they cannot be treated as protein sinks. Significantly higher levels of phenols were observed in the galled leaves and galls of all cynipid species when compared with the control tissues. Peroxidase and polyphenol oxidase activity was usually low or showed no activity in galled tissues of all species. PR proteins, such as chitinase and ß-1,3-glucanase, had a similar activity profile. Their activity significantly increased in the leaves with galls of all cynipid species, especially those infested with C. quercusfolii. Data generated in this study clearly indicate that galling Cynipidae manipulate the biochemical machinery of the galls for their own needs. However, the pattern of the biochemical features of leaves with galls and galled tissues depends on gall-making species.

Description, host range and distribution of a new Macrodiplosis species (Diptera: Cecidomyiidae) that induces leaf-margin fold galls on deciduous Quercus (Fagaceae) with comparative notes on Palaearctic congeners.

A gall midge that induces upwardly folded leaf-margin galls on Quercus serrata, Q. mongolica and Q. dentata (Fagaceae) in Japan and South Korea is described as Macrodiplosis selenis sp. n. (Diptera: Cecidomyiidae). M. selenis is distinguished from Palaearctic congeners by a combination of morphological characters. Genetic differences supported the result of morphological comparison and indicated that M. selenis is closely related to the European M. roboris, whose gall is similar to that of M. selenis.

Specific polyphenols and tannins are associated with defense against insect herbivores in the tropical oak Quercus oleoides.

The role of plant polyphenols as defenses against insect herbivores is controversial. We combined correlative field studies across three geographic regions (Northern Mexico, Southern Mexico, and Costa Rica) with induction experiments under controlled conditions to search for candidate compounds that might play a defensive role in the foliage of the tropical oak, Quercus oleoides. We quantified leaf damage caused by four herbivore guilds (chewers, skeletonizers, leaf miners, and gall forming insects) and analyzed the content of 18 polyphenols (including hydrolyzable tannins, flavan-3-ols, and flavonol glycosides) in the same set of leaves using high performance liquid chromatography and mass spectrometry. Foliar damage ranged from two to eight percent per region, and nearly 90% of all the damage was caused by chewing herbivores. Damage due to chewing herbivores was positively correlated with acutissimin B, catechin, and catechin dimer, and damage by mining herbivores was positively correlated with mongolinin A. By contrast, gall presence was negatively correlated with vescalagin and acutissimin B. By using redundancy analysis, we searched for the combinations of polyphenols that were associated to natural herbivory: the combination of mongolinin A and acutissimin B had the highest association to herbivory. In a common garden experiment with oak saplings, artificial damage increased the content of acutissimin B, mongolinin A, and vescalagin, whereas the content of catechin decreased. Specific polyphenols, either individually or in combination, rather than total polyphenols, were associated with standing leaf damage in this tropical oak. Future studies aimed at understanding the ecological role of polyphenols can use similar correlative studies to identify candidate compounds that could be used individually and in biologically meaningful combinations in tests with herbivores and pathogens.

Genetic characterization of a novel Iflavirus associated with vomiting disease in the Chinese oak silkmoth Antheraea pernyi.

Larvae of the Chinese oak silkmoth (Antheraea pernyi) are often affected by AVD (A. pernyi vomiting disease), whose causative agent has long been suspected to be a virus. In an unrelated project we discovered a novel positive sense single-stranded RNA virus that could reproduce AVD symptoms upon injection into healthy A. pernyi larvae. The genome of this virus is 10,163 nucleotides long, has a natural poly-A tail, and contains a single, large open reading frame flanked at the 5' and 3' ends by untranslated regions containing putative structural elements for replication and translation of the virus genome. The open reading frame is predicted to encode a 3036 amino acid polyprotein with four viral structural proteins (VP1-VP4) located in the N-terminal end and the non-structural proteins, including a helicase, RNA-dependent RNA polymerase and 3C-protease, located in the C-terminal end of the polyprotein. Putative 3C-protease and autolytic cleavage sites were identified for processing the polyprotein into functional units. The genome organization, amino acid sequence and phylogenetic analyses suggest that the virus is a novel species of the genus Iflavirus, with the proposed name of Antheraea pernyi Iflavirus (ApIV).

Interspecific patterns of phenotypic selection do not predict intraspecific patterns.

We estimated linear (ß) and nonlinear (γ) selection gradients to quantify host plant-mediated selection on the trait gall size in each of 22 unequally sampled subpopulations of the cynipid gall wasp Belonocnema treatae. We characterized the relationship between variation in subpopulation sample size and the magnitude of and the variance among selection gradients. We then tested the hypothesis that the intraspecific patterns we observed would follow two patterns that have emerged from published estimates of linear and nonlinear selection gradients compiled across species, namely that the average magnitude of ß and γ and the variance among estimated ß and γ decrease with increasing sample size. For both ß and γ, intraspecific patterns of phenotypic selection in relation to sample size were not predicted by interspecific patterns. Thus, our results suggest that when selection is heterogeneous among subpopulations, variation in the biological basis for selection is more important in influencing estimates of selection than is variation in study size. Our study highlights the value of inspecting selection in relation to sampling effort at the level at which understanding the sources of variation in selection is most important, among populations within species.

Parallel patterns of morphological and behavioral variation among host-associated populations of two gall wasp species.

A powerful approach to address the general factors contributing to ecological speciation is to compare distantly related taxa that inhabit the same selective environments. In this design, similarities among taxa can elucidate general mechanisms of the process whereas differences may uncover specific factors important to the process for individual taxa. Herein, we present evidence of parallel patterns of morphological and behavioral variation among host-associated populations of two species of cynipid gall wasps, Belonocnema treatae and Disholcaspis quercusvirens, that each exhibit a life cycle intimately tied to the same two host plant environments, Quercus geminata and Q. virginiana. Across both gall-former species we find consistent differences in body size and gall morphology associated with host plant use, as well as strong differences in host plant preference, a measure of habitat isolation among populations. These consistent differences among taxa highlight the important role of host plant use in promoting reproductive isolation and morphological variation among herbivorous insect populations-a prerequisite for ecological speciation.

A new genus of oak gallwasp, Cyclocynips Melika, Tang & Sinclair (Hymenoptera: Cynipidae: Cynipini), with descriptions of two new species from Taiwan.

A new genus of cynipid oak gallwasp-Cyclocynips Melika, Tang, & Sinclair (Hymenoptera: Cynipidae: Cynipini), with two new species--C. uberis and C. tumorvirgae--reared from galls on oaks of the Quercus subgenus Cyclobalanopsis is described from Taiwan. Descriptions of asexual generation adults and their diagnostic characters are presented. The likelihood of yet undiscovered sexual generations and the evolution of host-plant associations in these species are discussed.

Catalogue of parasitoids and inquilines in cynipid oak galls in the West Palaearctic.

A quantitative catalogue of the parasitoids (almost exclusively Chalcidoidea) and inquiline Cynipidae recorded in the western Palaearctic from galls induced on Quercus by Cynipidae (Cynipini) is presented. Quantitative and national data are included with bibliographic references to almost all records published in 2011 and earlier. The catalogue is followed by two checklists, firstly one of the Chalcidoidea with numbers of each species recorded from each type of host gall (galls of the sexual and asexual generations of the host gall wasps are listed separately), and secondly one of inquiline Cynipidae with host galls. Compared to non-oak gall wasps, the Cynipini support a much larger parasitoid and especially inquiline fauna, and this fauna is very largely restricted at the species level to Cynipini galls. About one hundred chalcidoid species are recorded from galls of Cynipini, distributed over six families: Pteromalidae and Eulophidae (29 species each), Torymi-dae (21 species), Eurytomidae (10 species), Eupelmidae (8 species) and Ormyridae (at least 2 species). Polyphagy is usual in the chalcidoid parasitoids, most species having a broad host gall range, but quantitatively the fauna of each type of oak gall is rather characteristic and is strongly influenced by gall morphology, situation on the tree, season of growth and host tree species. These and other extrinsic factors restrict the full exploitation of the chalcidoids' potential host gall range.

Function of defensive volatiles in pedunculate oak (Quercus robur) is tricked by the moth Tortrix viridana.

The indirect defences of plants are comprised of herbivore-induced plant volatiles (HIPVs) that among other things attract the natural enemies of insects. However, the actual extent of the benefits of HIPV emissions in complex co-evolved plant-herbivore systems is only poorly understood. The observation that a few Quercus robur L. trees constantly tolerated (T-oaks) infestation by a major pest of oaks (Tortrix viridana L.), compared with heavily defoliated trees (susceptible: S-oaks), lead us to a combined biochemical and behavioural study. We used these evidently different phenotypes to analyse whether the resistance of T-oaks to the herbivore was dependent on the amount and scent of HIPVs and/or differences in non-volatile polyphenolic leaf constituents (as quercetin-, kaempferol- and flavonol glycosides). In addition to non-volatile metabolic differences, typically defensive HIPV emissions differed between S-oaks and T-oaks. Female moths were attracted by the blend of HIPVs from S-oaks, showing significantly higher amounts of (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E)-ß-ocimene and avoid T-oaks with relative high fraction of the sesquiterpenes α-farnesene and germacrene D. Hence, the strategy of T-oaks exhibiting directly herbivore-repellent HIPV emissions instead of high emissions of predator-attracting HIPVs of the S-oaks appears to be the better mechanism for avoiding defoliation.