Sexually dimorphic gall structures correspond to differential phytohormone contents in male and female wasp larvae

Sexually dimorphic galls are rare among gall‐inducing insects and the reason for their occurrence is unknown. The pteromalid wasp Trichilogaster acaciaelongifoliae, which induces galls on Acacia longifolia, is one such species. In the present study, the anatomical and physiological attributes of male and female galls of T. acaciaelongifoliae are examined and compared. Histological preparations are used to characterize anatomical differences between male and female gall chambers. Bioassays, high‐performance liquid chromatography‐mass spectrometry and an enzyme immunoassay are used to measure concentrations of auxin and cytokinin in normal buds, galled tissues, and larvae of both sexes. Female chambers are found to be 3.3‐fold larger, and are associated with 1.5‐fold more storage tissue and 3.5‐fold more vascular tissues than male chambers. Tissues from female chambers induce stronger cytokinin‐like bioactivity than tissues from male chambers. Female larvae have considerably higher concentrations of cytokinin free bases, ribosides, glucosides and monophosphates than male larvae; higher auxin‐like bioactivity than in normal or galled plant tissues; and almost twice the concentration of auxin than male larvae. Both male and female larvae contain much higher auxin concentrations than either galled or normal plant tissues. These findings suggest that differing levels of phytohormones are involved in the development of sexual dimorphism of gall structures in this species.     

Transcriptomic characterization of gall tissue of Japanese elm tree (Ulmus davidiana var. japonica) induced by the aphid Tetraneura nigriabdominalis

Insect galls are abnormal plant tissues induced by parasitic insect(s) for use as their habitat. In previous work, we suggested that gall tissues induced by the aphid Tetraneura nigriabdominalis on Japanese elm trees are less responsive than leaf tissues to jasmonic acid (JA), which is involved in the production of volatile organic compounds as a typical defensive reaction of plants against attack by insect pests. A comprehensive analysis of gene expression by RNA sequencing indicated that the number of JA responsive genes was markedly lower in gall tissues than in leaf tissues. This suggests that gall tissues are mostly defective in JA signaling, although JA signaling is not entirely compromised in gall tissue. Gene ontology analysis sheds light on some stress-related unigenes with higher expression levels in gall tissues, suggesting that host plants sense aphids as a biotic stress but are defective in the JA-mediated defense response in gall tissues.     

Anti-insect effects of the gall wall of Baizongia pistaciae [L.], a gall-inducing aphid on Pistacia palaestina Boiss

The Enemy hypothesis is a theoretical framework for understanding the adaptive nature of galls induced in host plants by insects. Contrary to other gall inducing insects, like Cynipids or sawflies, this hypothesis has not been studied for the gall aphids on pistachio trees in the Middle East. Galls on plants are supposed to protect their inducers from other organisms, including herbivores feeding on the host plant and possibly feeding on the gall tissue. Assuming that among aphid enemies there are numerous insects which have to perforate the gall wall to access the aphids inside, determining whether the gall wall has anti-insect properties should be one of the first steps in dealing with this hypothesis. In the present research using Baizongia pistaciae [L.], an aphid that creates perfectly closed galls in Pistacia palaestina Boiss, laboratory experiments were first conducted on a herbivore, the stored grain pest, Tribolium castaneum Herbst, to assess chemical anti-insect activities of the gall tissue, and an effort was made to understand why these properties do not harm the aphids inside the gall. Addition of fresh gall tissue to food reduced the population growth of flour beetles. Non-polar organic extracts had contact toxicity for larvae of these insects, and an impact on the feeding preferences of the adults. These results indicate chemical anti-insect activities of the gall tissue. The research also reveals that the permeability of the gall wall to non-polar volatile compounds is important to the survival of the aphids inside the gall cavity. These findings do not allow us to reject the Enemy hypothesis in the gall-inducing aphids/Pistacia trees interactions.     

Manipulation of host plant cells and tissues by gall-inducing insects and adaptive strategies used by different feeding guilds

Biologists who study insect-induced plant galls are faced with the overwhelming diversity of plant forms and insect species. A challenge is to find common themes amidst this diversity. We discuss common themes that have emerged from our cytological and histochemical studies of diverse neotropical insect-induced galls. Gall initiation begins with recognition of reactive plant tissues by gall inducers, with subsequent feeding and/or oviposition triggering a cascade of events. Besides, to induce the gall structure insects have to synchronize their life cycle with plant host phenology. We predict that reactive oxygen species (ROS) play a role in gall induction, development and histochemical gradient formation. Controlled levels of ROS mediate the accumulation of (poly)phenols, and phytohormones (such as auxin) at gall sites, which contributes to the new cell developmental pathways and biochemical alterations that lead to gall formation. The classical idea of an insect-induced gall is a chamber lined with a nutritive tissue that is occupied by an insect that directly harvests nutrients from nutritive cells via its mouthparts, which function mechanically and/or as a delivery system for salivary secretions. By studying diverse gall-inducing insects we have discovered that insects with needle-like sucking mouthparts may also induce a nutritive tissue, whose nutrients are indirectly harvested as the gall-inducing insects feeds on adjacent vascular tissues. Activity of carbohydrate-related enzymes across diverse galls corroborates this hypothesis. Our research points to the importance of cytological and histochemical studies for elucidating mechanisms of induced susceptibility and induced resistance.     

Crown gall disease and hairy root disease : a sledgehammer and a tackhammer

The neoplastic diseases crown gall and hairy root are incited by the phytopathogenic bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Although the molecular mechanism of T-DNA transfer to the plant most likely is the same for both species, the physiological basis of tumorigenesis is fundamentally different. Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin specified by A. tumefaciens T-DNA genes. Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. An understanding of hairy root tumorigenesis will likely result in an increased knowledge of plant developmental processes.     

Crown-gall-resistant transgenic apple trees that Silence Agrobacterium tumefaciens oncogenes

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.these authors contributed equally to this workthese authors contributed equally to this work     

Morphometric analysis of young petiole galls on the narrow-leaf cottonwood, <Emphasis Type="Italic">Populus angustifolia</Emphasis>, by the sugarbeet root aphid, <Emphasis Type="Italic">Pemphigus betae</Emphasis>

An insect-induced gall is a highly specialized structure resulting from atypical development of plant tissue induced by a reaction to the presence and activity of an insect. The insect induces a differentiation of tissues with features and functions of an ectopic organ, providing nutrition and protection to the galling insect from natural enemies and environmental stresses. In this anatomical and cytological study, we characterized how the gall-inducing aphid Pemphigus betae reshapes the leaf morphology of the narrow-leaf cottonwood Populus angustifolia to form a leaf fold gall. Young galls displayed a bend on one side of the midvein toward the center of the leaf and back to create a fold on the abaxial side of the leaf. This fold was formed abaxially by periclinal and anticlinal divisions, effectively eliminating intercellular spaces from the spongy parenchyma. Galls at this stage exhibited both cell hypertrophy and tissue hyperplasia. Cells on the adaxial surface were more numerous and smaller than cells near the abaxial surface were, creating the large fold that surrounds the insect. Mesophyll cells exhibited some features typical of nutritive cells induced by other galling insects, including conspicuous nucleolus, reduced and fragmented vacuole, smaller and degraded chloroplasts, and dense cytoplasm compared to ungalled tissue. Even though aphids feed on the contents of phloem and do not directly consume the gall tissue, they induce changes in the plant vascular system, which lead to nutrient accumulation to support the growing aphid numbers in mature galls.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

A galling aphid furnishes its home with a built-in pipeline to the host food supply

Galling insects modify the developmental pathways of their host plants and create a protected and favourable microhabitat for their offspring. Galling aphids reproduce in their galls and the resulting clones often comprise hundreds of individuals. We followed the histological changes in the host Pistacia palaestina (Anacardiaceae) induced by the galling aphid Geoica wertheimae (Aphidoidea, Pemphigidae: Fordinae). We showed that the leaf tissues are altered in the gall in a way that gives the aphids easy access to the vascular system from which they obtain their nourishment. Specifically, the cuticle that lines the normal leaf epidermal cells is absent on the surface of the inner gall cavity, and the normal palisade cells are replaced by parenchymatous tissue with numerous wide latex ducts. Associated with these ducts, many new sieve tubes (phloem elements) are formed just a few cell layers below the inner gall surface. This arrangement enables the hundreds of aphids to feed simultaneously and continuously in the gall throughout the summer.     

Exogenous phytohormones and the induction of plant galls by insects

The mechanism of gall induction by insects has remained elusive. Previous studies have met with limited success in attempting to induce galls by injection or application of chemical compounds. To determine whether an exogenous source of phytohormones plays a role in gall induction, we injected cytokinin (CK), auxin (IAA), gibberellic acid (GA), and abscisic acid (ABA) in various concentrations, ratios, and combinations into leaf petioles of Capsicum annuum L. cv. California Wonder (Solanaceae). We found that CK + IAA injections lead to gall-like growth in C. annuum. GA enhanced and ABA inhibited gall growth except in the presence of GA. Isopentenyl adenine (IP) was the most effective type of CK at inducing growth. Our work is consistent with the hypothesis that exogenous CK + IAA produced and supplied by insects leads to gall induction. We hypothesize that insects have obtained the capability to induce galls via acquisition of the biosynthetic pathways to produce IAA and trans-zeatin family CKs through microbial symbiosis or lateral gene transfer.     

Temperature-sensitive loss of tumor characteristics in a tobacco crown gall strain

Summary Three independently isolated tobacco crown gall strains incited byAgrobacterium tumefaciens C58 required phytohormone (auxin and cytokinin) supplements in the basal medium to grow, at 37°C. Six other tobacco crown gall strains incited, respectively, byA. tumefaciens IIBV7, B6, CGIC, A6NC, 27 and AT4 expressed, at 37°C, the tumor characteristic of ability to grow in vitro on medium lacking phytohormones. Nopaline was not detectable in C58 tumors cultured at 37°C, but octopine was produced by B6 tumor tissues incibated at the elevated temperature. C58 tumor strains kept at 37°C for 1 week or more lost the ability to express tumor characteristics at 27°C such as tissue morphology, growth on basal medium lacking phytohormones and nopaline production. Heat-treated C58 tissues also differed from the original tumor strain in regeneration ability and phytohormone requirements of explants; i.e. explants from regenerated, heart-treated C58 tumors required both auxin and cytokinin for growth in vitro.     

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.     

The mechanism of gall induction makes galls red

We propose that the commonly observed red coloration of insect-induced plant galls is due to the production of exogenous cytokinins by gall-inducing insects. A growing body of evidence indicates that gall-inducing insects, bacteria, and fungi produce cytokinins. We hypothesize that gall induction generally requires an exogenous source of cytokinin and auxin. Plant galls are mobilizing sinks induced by cytokinin and reinforced by transport and accumulation of sugar. Exogenous cytokinins lead to a cascade of effects including the up-regulation of anthocyanin synthesis, the source of red coloration. Experiments demonstrate that exogenous cytokinins and sugars up-regulate the phenylpropanoid and flavonoid pathways, leading to localized anthocyanin accumulation. We suggest that red coloration in plant galls is merely a consequence of the mechanism of gall induction, and therefore an example of fabricational noise rather than aposematic coloration. Only color manipulation experiments can determine whether gall color is also secondarily aposematic.     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

Synchronization of gallers with host plant phenology

In addition to various bottom-up effects, the synchronization of herbivores with their host plant phenology determines quality and quantity of food resources and affects the preference–performance linkage and abundance of herbivores. The synchronization has a more critical meaning for such short-lived galling insects as cecidomyiid adults and young aphid stem mothers than for other insects. This review, first, presents general information about gall midges and gall aphids, together with their life history patterns and some ecological attributes. Second, some important topics of galling insect–host plant relation are briefly reviewed. Then, synchronization patterns between gall midge emergence and host plant phenology are analyzed to discuss the adaptive strategies of gall midges and to show how the amount of available food resources is affected by the time lag in synchronization. The spatial distribution pattern and the preference–performance linkage of aphid stem mothers is also discussed in relation to synchronization. Received: October 2, 1998 / Accepted: July 3, 2000     

Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density

Trehalose is a disaccharide sugar that is now considered to be widely distributed among higher plants. Trehalose has been attributed a number of roles, including control of basic plant processes, such as photosynthesis, and conferring tolerance to abiotic stresses, such as desiccation and high salinity. Trehalose is also a common storage sugar used by insects. In this study, we used laboratory investigations to examine various aspects of trehalose dynamics in an aphid–host plant system (Arabidopsis and the peach potato aphid, Myzus persicae). Trehalose concentrations were measured by [1-H]-NMR. Myzus persicae reared on Arabidopsis, but not on black mustard or spring cabbage, contained considerable quantities of trehalose (5 % w/w dry matter). In Arabidopsis foliage, feeding by aphids induced a density-dependent accumulation of trehalose up to 5 mg g^?1 dry weight. Leaves that were not challenged directly by aphids also exhibited increased trehalose concentrations, indicating that this accumulation was systemic. Trehalose was measured at high concentrations in the phloem sap of plants challenged by aphids, suggesting that aphid feeding induced the plant to produce significant quantities of trehalose, which moved through the plant and into the aphids via the phloem sap. Trehalose was also excreted in the aphid honeydew. Further work is required to clarify whether this trehalose accumulation in Arabidopsis has a direct role or a signalling function in plant tolerance of, or resistance to, aphid feeding, and if a similar accumulation of this sugar occurs when other species or genotypes of aphids are reared on this host plant.