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.     

Competition and niche relationships among Eriosoma aphids occurring on the Japanese elm

Summary Galls of more than one species of Eriosoma (Aphidoidea) are found sympatrically even on single trees. Incipient galls are frequently invaded by conspecific and/or allospecific fundatrices. Eriosoma yangi, a component of Eriosoma communities, does not form its own galls but obligatorily usurps those of other species. There were interspecific differences in the timing of gall formation and the spatial distribution of galls. Nevertheless E. yangi fundatrices randomly invaded galls of any Eriosoma species and occupied 33%–41% of galls of each species. When more than one E. yangi fundatrix invaded one gall, mortal fights sometimes occurred. Fundatrices of gall-forming species also seemed to take part in such fights. Fundatrices of gall-forming species had a significant tendency to invade galls of a particular species. However, taking account of niche differences among species, invaders apparently entered available galls at random. Apparently E. yangi fundatrices search an extensive range within a branch for galls, while invaders of gall-forming species search a restricted speciesspecific range. The niche relation of gall-forming species in a northern community containing E. yangi were compared with those in a southern community lacking E. yangi. No obvious difference was found between them, suggesting that parasitism by E. yangi has not influenced niche divergence within the Eriosoma community.     

Hormaphis hamamelidis and gall size: a test of the plant vigor hypothesis

We tested the Plant Vigor Hypothesis by determining the distribution of galls formed on leaves of witch hazel, Hamamelis virginiana , by the aphid Hormaphis hamamelidis , and by determining various factors that affect the fecundity of the gall-forming fundatrices. We also studied the role of the fundatrix in host plant manipulation. While the mean number of galls per leaf was low, galls had an aggregated distribution among leaves. Among trees, the average number of galls per leaf was not related to the mean leaf size, contrary to the preference prediction of the Plant Vigor Hypothesis. While fundatrices preferred the distal leaves of buds, which grew more than the proximal leaves, being on distal leaves conferred no increase in fecundity for fundatrices, contrary to predictions of the Plant Vigor Hypothesis. Gall size was the factor that explained the largest proportion of variation in fundatrix fecundity; fundatrix size explained somewhat less of the variation. Also, gall position on the leaf, number of aphid galls on the leaf, and on which leaf of the bud the gall was located all played small, statistically significant roles in explaining fundatrix fecundity, but their effects were variable between experiments. Removal of fundatrices shortly after galls had enclosed them limited the growth of galls, indicating the role of the fundatrices in gall growth. We compare and contrast this system versus other gall-forming insects, as well as discuss the adaptive significance of the aphid manipulation of the host plant. Much of the data contradict predictions of the Plant Vigor Hypothesis, and we discuss how gall size, as a measure of plant growth caused by insect manipulation, explains the observed patterns.     

Aphid galls as a double food source for woodpeckers

An adult female and a young great spotted woodpecker Dendrocopos major were found exploiting galls of the elm balloon‐gall aphid Eriosoma lanuginosum by eating both the insects and their honeydew. Literature offers sparse and indirect information to explain this behavior. Taking both aphids and honeydew from within galls is reported here for the first time for a bird species. Only one other vertebrate, the Eurasian red squirrel Sciurus vulgaris, would show similar behavior. In contrast with the seeming rarity of such a feeding way, likely advantages in the double food source suggest it may have been overlooked.     

Maternal death relaxes developmental inhibition in nymphal aphid defenders

In certain aphids, first-instar nymphs defend their gall by attacking intruding arthropod predators. One correlate of such defensive behaviour is a lengthened duration of the first nymphal stadium during the galling phase of the life cycle. A prolonged first stadium allows a large army of first-instar defenders to accumulate, which may be advantageous for gall defence. The factors determining developmental delay have been unclear, however. Our field experiment with Pemphigus obesinymphae, a North American gall-forming aphid with defensive first-instar nymphs, tests whether first-stadium duration is influenced by the death of the colony''s fundatrix (mother). We killed fundatrices in certain galls, left those in control galls alive, and counted aphids in each stadium in each gall. Galls in which fundatrices were killed contained a lower proportion of first-instar defenders and more late-instar nymphs than did galls with living fundatrices, indicating that maternal death dramatically increased developmental rate of nymphs. Possibly nymphal aphids respond adaptively to environmental cues that signal a threat to the colony''s welfare. Alternatively, the fundatrix actively suppresses offspring development in order to maintain a large army of soldiers to protect her gall. The results add a new layer of complexity to our understanding of social aphid systems.     

Local adaptation and host race formation of a gall-forming aphid in relation to environmental heterogeneity

Summary The process of host race formation in the aphid Tetraneura yezoensis is examined in relation to its population structure. T. yezoensis induces pouch galls on new leaves of Ulmus davidiana and U. laciniata. Its populations on the two host species are often sympatric. Fundatrices found on one elm species, when reciprocally transplanted to the other, suffered greatly reduced average fitness. This shows that aphid populations associated with the two elm species are genetically differentiated in physiological traits. Individual trees of each elm species showed large differences in susceptibility to gall formation and in bud burst time, and such between-tree variations were consistent over years. Overwintered eggs taken in early spring from four trees (two from each species) were incubated under the same temperature conditions. The average hatching time differed significantly even between populations from conspecific trees, and the sequence of egg hatching paralleled that of the leafing of those four trees. This between-tree difference in hatching time was consistent over years and was found to be genetic, showing that gene flow between aphid populations on separate trees is often restricted. The heterogeneity in host traits may have promoted the evolution of philopatry in this aphid. Of the fundatrices that hatched on a tree of one elm species, a few precent were preadapted to gall formation on the other elm species. This suggests that the formation of a new host race proceeds parapatrically under disruptive selection and at a low level of gene flow. Evidence was actually obtained that a small fraction of Tetraneura alates are passively transported and land on non-host plants.     

The evolution of inquilinism, host-plant use and mitochondrial substitution rates in Tamalia gall aphids

We used mitochondrial DNA data to infer phylogenies for 28 samples of gall-inducing Tamalia aphids from 12 host-plant species, and for 17 samples of Tamalia inquilinus, aphid 'inquilines' that obligately inhabit galls of the gall inducers and do not form their own galls. Our phylogenetic analyses indicate that the inquilines are monophyletic and closely related to their host aphids. Tamalia coweni aphids from different host plants were, with one exception, very closely related to one another. By contrast, the T. inquilinus aphids were strongly genetically differentiated among most of their host plants. Comparison of branch lengths between the T. coweni clade and the T. inquilinus clade indicates that the T. inquilinus lineage evolves 2.5-3 times faster for the cytochrome oxidase I gene. These results demonstrate that: (1) Tamalia inquilines originated from their gall-inducing hosts, (2) communal (multi-female) gall induction apparently facilitated the origin of inquilinism, (3) diversification of the inquilines has involved rapid speciation along host-plant lines, or the rapid evolution of host-plant races, and (4) the inquilines have undergone accelerated molecular evolution relative to their hosts, probably due to reduced effective population sizes. Our findings provide insight into the behavioural causes and evolutionary consequences of transitions from resource generation to resource exploitation.     

Gall distribution as a compromise between the optimal gall-site selection and the synchrony to host-plant phenology in the aphid Kaltenbachiella japonica

Gall-site selection by the aphid Kaltenbachiella japonica was evaluated in relation to leaf position in a shoot, and gall positions within a leaf. First-instar fundatrices induce closed galls on the midribs of host leaves, and several galls were often induced on one leaf. Leaves with many galls were often withered before emergence of sexuparae from the galls. Within a leaf, gall volume was positively correlated with the sum of lateral-vein length in the leaf segment at which the gall was induced. The observed pattern in gall volume among the leaf segments corresponded with that in the lateral-vein length. These results show that a foundatrix selects the most vigorous position within a leaf to produce more offspring. Although distal leaves grew faster than did basal leaves, gall density was highest on leaves at the middle order when a shoot has more than seven leaves. Optimal gall-site selection seems to be constrained by the asynchrony in timing between the hatching of fundatrices and leaf growth within a shoot. These results suggest that the observed gall distribution is affected by both the distribution of suitable galling sites within a leaf and the synchrony with leaf phenology of the host plant.     

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.     

Aphid galls accumulate high concentrations of amino acids: a support for the nutrition hypothesis for gall formation

The nutrition hypothesis for the adaptive significance of insect gall formation postulates that galls accumulate higher concentrations of nutritive compounds than uninfested plant tissue, resulting in a high performance of the gall former. This hypothesis has been supported by some taxa of gall insects, but not by taxa such as cynipid wasps. Aphid galls are expected to require higher levels of nitrogen than other insects’ galls with a single inhabitant, because aphid galls are required to sustain a number of aphids reproducing parthenogenetically over two generations. The present study tested this hypothesis by evaluating aphid performance and amino acid concentration in phloem sap, using the aphid Rhopalosiphum insertum (Walker) (Homoptera: Aphididae), which establishes colonies on leaves of Sorbus commixta Hedlund or in galls of the aphid Sorbaphis chaetosiphon Shaposhnikov (Homoptera: Aphididae). We prepared the gall and non‐gall treatments on trees of S. commixta, in which R. insertum fundatrices were reared and allowed to reproduce. In S. chaetosiphon galls, R. insertum colonies propagated more rapidly, and the second generation grew larger and more fecund than on ungalled leaves. The amount of amino acids exuding from cut galled leaves was fivefold that in ungalled leaves; however, there was no significant difference in the amino acid composition between galled and ungalled leaves. In the intact leaves, total amino acid concentration in the phloem sap declined rapidly from late April to late May; however, the galls retained this high amino acid concentration in developing leaves for 1 month. These results indicate that the improved performance in R. insertum is ascribed to the increased concentration of amino acids in galled leaves. We suggest that S. chaetosiphon galls function to promote the breakdown of leaf protein, leading to an increased performance of gall‐inhabiting aphids.     

Trade‐off between fecundity and survival generates stabilizing selection on gall size

Complex interactions within multitrophic communities are fundamental to the evolution of individual species that reside within them. One common outcome of species interactions are fitness trade‐offs, where traits adaptive in some circumstances are maladaptive in others. Here, we identify a fitness trade‐off between fecundity and survival in the cynipid wasp Callirhytis quercusbatatoides that induces multichambered galls on the stem of its host plant Quercus virginiana. We first quantified this trade‐off in natural populations by documenting two relationships: a positive association between the trait gall size and fecundity, as larger galls contain more offspring, and a negative association between gall size and survival, as larger galls are attacked by birds at a higher rate. Next, we performed a field‐based experimental evolution study where birds were excluded from the entire canopy of 11 large host trees for five years. As a result of the five‐year release from avian predators, we observed a significant shift to larger galls per tree. Overall, our study demonstrates how two opposing forces of selection can generate stabilizing selection on a critical phenotypic trait in wild populations, and how traits can evolve rapidly in the predicted direction when conditions change.     

Coexistence and weak amensalism of congeneric gall-forming aphids on the Japanese elm

Closely related species of gall-forming aphids are often associated with a single host species. SixTetraneura species coexist on the Japanese elm,Ulmus davidiana, in Sapporo, northern Japan. This paper describes the probabilities of coexistence on macro- and microgeographic scales (i.e., on host trees and host leaves) and examines whether coexistence with conspecific or heterospecific galls on leaves or shoots has any effect on the fecundity of each aphid species using multiple regression. A Monte Carlo simulation showed that the frequency distribution of the numbers of species on individual host trees differed significantly from that expected from the null model. There were significantly positive or negative associations between species.Tetraneura radicicola andsorini always coexisted with other species on trees they infested. Multiple regression revealed that the coexistence of conspecific or heterospecific galls on individual leaves had no influence on the fecundity ofradicicola andsorini, but had a negative influence on that of sp. O. On average,radicicola andsorini produced a smaller number of offspring in galls than sp. O, and obviously consumed only a small part of resources available on the galled leaves. Evidence available suggests that although amensalism does arise between sp. O and other species, its influence in not so strong as to exclude sp. O competitively from theTetraneura community.     

Clonal mixing in the soldier-producing aphid Pemphigus spyrothecae (Hemiptera: Aphididae)

Illuminating the genetic relationships within soldier-producing aphid colonies is an essential element of any attempt to explain the evolution of the altruistic soldier caste. Pemphigus spyrothecae is a soldier-producing aphid that induces galls on the leaf petioles of its host (trees of the genus Populus). At least a quarter of the aphids within the clonally produced gall population are morphologically and behaviourally distinct first-instar soldiers that defend the gall population from predation. Using field trapping and microsatellites, we investigated the degree of clonal mixing within natural gall populations. Field trapping in the UK showed that all the migrants of P. spyrothecae and of two other Pemphigus species were wingless first-instar soldiers. The average degree of mixing estimated from trapping P. spyrothecae migrants was 0.68% (range = 0-15%). Microsatellite genotyping of 277 aphids from 13 galls collected in Italy revealed an average mixing level of 10.4% (range = 0-59%). Six galls contained more than one clone (range = 2-5 clones). Non-kin aphids were not restricted to the soldier caste but were evenly distributed across instars. An additional gall, from which 527 occupants were genotyped, contained 12 non-kin aphids distributed among nine clones, showing that clonal diversity can be high even when mixing is very low. These observations suggest that although soldiers migrate regularly and can moult and reproduce within foreign galls, clonal mixing in this species is generally low and is unlikely to provide a barrier to the evolution of investment by the aphid clones in an altruistic soldier caste.     

Phylogeny of Rhus gall aphids (Hemiptera : Pemphigidae) based on combined molecular analysis of nuclear EF1α and mitochondrial COII genes

Rhus gall aphids (Fordinae : Melaphidini) have a disjunct distribution in East Asia and North America and have specific host plant relationships. Some of them are of economic importance and all species form sealed galls which show great variation in shape, size, structure, and galling‐site. We present a phylogeny incorporating ten species and four subspecies of Rhus gall aphids based on 1694 base pairs of nuclear elongation factor‐1α (EF1α) and mitochondrial cytochrome oxidase subunit II (COII) DNA sequence data. The results suggest that Melaphidini is monophyletic and at the genus level, Schlechtendalia, Nurudea, and Floraphis were each monophyletic. Kaburagia and Meitanaphis were not monophyletic and therefore inconsistent with the current classification. The North American sumac gall aphid, Melaphis rhois, was most closely related to the East Asian Floraphis species, although this was poorly supported. The conservation of gall morphology with respect to aphid phylogeny rather than their host plants suggests that gall morphology is largely determined by the aphids. While there is no evidence of strict co‐speciation between the aphids and their primary host plants, switching between recently diverged host plants may be involved in the speciation process in Melaphidini.     

High mean relatedness among communally galling Tamalia aphids revealed by AFLP analysis

Cooperative or eusocial behavior occurs in gall-inducing insects, but the ecological and evolutionary contexts for these behaviors vary in their details. Foundresses of the manzanita leaf-gall aphid, Tamalia coweni, regularly share galls. Because aphids undergo parthenogenesis, communal foundresses are potential clone mates. Under the kin selection hypothesis, a high level of relatedness is predicted to lower genetically based conflict among females in a group and thereby favor communal gall occupation. We recorded the frequency of communal behavior in a population of T. coweni on its host plant, Arctostaphylos patula, and measured reproductive output in single- and multiple-occupant galls. Eleven percent of the 375 galls examined were communally occupied, with double-foundress galls the commonest class, up to a maximum of five foundresses within galls. Total productivity of communal galls (measured by numbers of offspring per gall) was higher than for single-foundress galls on a per-gall basis, but lower per capita. We genotyped foundresses with amplified fragment length polymorphism (AFLP)-PCR markers, to estimate relatedness among gall co-occupants and foundresses from randomly selected galls in the population. Analysis of genetic distance between communal foundresses revealed that relatedness among gall cohabitants was significantly higher, on average, than for foundresses drawn from the population at random (P < 0.001). Phylogenetic analysis using parsimony (PAUP) of the AFLP profiles indicated that half the foundresses sharing galls were most closely related to their gall mates. Our results are consistent with kin selection theory, and suggest that communal gall occupation in this species may be interpreted as cooperative behavior.     

Evolution of the gall wasp-host plant association

Gall wasps, or cynipids, form the second largest radiation of galling insects with more than 1300 described species. According to current views, the first cynipids were phytophagous and developed in herb stems of the Asteraceae without modifying plant growth or development. The first galls were supposedly multichambered stem swellings, and subsequent trends involved increase in gall complexity and reduction in the number of larval chambers. Gall wasps also have many of the features believed to be characteristic for phytophagous insects radiating in parallel with their host plants. We tested these hypotheses by mapping characters onto a recent estimate of higher cynipid relationships from a morphology-based analysis of exemplar taxa, controlling for phylogenetic uncertainty using bootstrapping. Characters were also mapped onto a metatree including all gall wasps, assembled from phylogenetic analyses as well as recent classifications. The results contradict many of the current hypotheses. The first cynipids with extant descendants were not Asteraceae stem feeders but induced distinct single-chambered galls in reproductive organs of herbaceous Papaveraceae, or possibly Lamiaceae. There has been a general trend toward more complex galls but the herb-stem feeders evolved from ancestors inducing distinct galls and their larval chambers are best understood as cryptic galls. Woody hosts have been colonized only three times, making the apparently irreversible transition from herbs to woody hosts one of the most conservative features of the gall wasp-host plant association. The evolution of host plant preferences is characterized by colonization of preexisting host-plant lineages rather than by parallel cladogenesis. Cynipids are mono- or oligophagous and host-plant choice is strongly phylogenetically conserved. Yet, the few major host shifts have involved remarkably distantly related plants. Many shifts have been onto plant species already exploited by other gall wasps, suggesting that interspecific parasitism among cynipids facilitates colonization of novel host plants.     

Partitioning of herbivore hosts across time and food plants promotes diversification in the Megastigmus dorsalis oak gall parasitoid complex

Communities of insect herbivores and their natural enemies are rich and ecologically crucial components of terrestrial biodiversity. Understanding the processes that promote their origin and maintenance is thus of considerable interest. One major proposed mechanism is ecological speciation through host‐associated differentiation (HAD), the divergence of a polyphagous species first into ecological host races and eventually into more specialized daughter species. The rich chalcid parasitoid communities attacking cynipid oak gall wasp hosts are structured by multiple host traits, including food plant taxon, host gall phenology, and gall structure. Here, we ask whether the same traits structure genetic diversity within supposedly generalist parasitoid morphospecies. We use mitochondrial DNA sequences and microsatellite genotypes to quantify HAD for Megastigmus (Bootanomyia) dorsalis, a complex of two apparently generalist cryptic parasitoid species attacking oak galls. Ancient Balkan refugial populations showed phenological separation between the cryptic species, one primarily attacking spring galls, and the other mainly attacking autumn galls. The spring species also contained host races specializing on galls developing on different host‐plant lineages (sections Cerris vs. Quercus) within the oak genus Quercus. These results indicate more significant host‐associated structuring within oak gall parasitoid communities than previously thought and support ecological theory predicting the evolution of specialist lineages within generalist parasitoids. In contrast, UK populations of the autumn cryptic species associated with both native and recently invading oak gall wasps showed no evidence of population differentiation, implying rapid recruitment of native parasitoid populations onto invading hosts, and hence potential for natural biological control. This is of significance given recent rapid range expansion of the economically damaging chestnut gall wasp, Dryocosmus kuriphilus, in Europe.     

Plant module size and dose of gall induction stimulus influence gall induction and galler performance

Several different hypotheses attribute large variations in gall abundance and galler performance to variations in plant growth rate and/or module size. The plant vigor hypothesis (PVH) predicts that galler performance will be greatest on large, fast‐growing plant modules due to their large, rapidly dividing cells that allow them to make large galls, where survival and size of survivors are usually greater. The optimal module size hypothesis (OMSH) predicts that galler performance will be greatest on intermediate‐sized modules, based on a tradeoff between ease of gall induction on small modules and increased resource availability in large modules. Here, we evaluate predictions of the PVH and OMSH during a two‐year field study by examining gall induction and full gall development success, as well as subsequent galler performance in successfully developed galls, using a shoot galling adelgid, Adelges abietis, on white spruce Picea glauca. In addition, we tested whether increased dose of gall induction stimulus on different sized modules, achieved by observing differing A. abietis densities per bud, could increase the rate of successful gall induction, as predicted by the OMSH. Galls of A. abietis appeared to be induced by a dose‐dependent stimulus from fundatrices in spring. Furthermore, the critical stimulus dose required to induce a gall appeared to increase with shoot length. These findings support the OMSH and not the PVH. Galler performance (gall volume and the number of gallicolae that emerged from each gall) in successfully developed galls was positively related to shoot length, supporting both the PVH and OMSH. We conclude that the PVH represents one component of the more inclusive OMSH, which considers both ease of gall induction on different‐sized modules and the effect of plant module size on gall size.     

The impact of kleptoparasitic invasions on the evolution of gall-size in social and solitary Australian Acacia thrips

Many species of gall-inducing Acacia thrips are attacked by kleptoparasitic thrips who enter the gall, destroy the occupants, and then use the gall for producing their own offspring. The hypothesis tested here is that pressure exerted by ldeptoparasites （genus Koptothrips） not only provoked the evolution of soldiers in the gall-inducing clade, but have also influenced the evolution of gall size and morphology. Various size dimensions of invaded galls were compared to those of uninvaded galls using data from six gall-inducing species and their kleptoparasites. For the non-social gall-inducing species （K. ellobus and K. nicholsoni） invaded galls showed no significant size differences from galls that had not been invaded. For the four social gall-inducingspecies （K. habrus, K. intermedius, K. waterhousei and K. morrisi） invaded galls were significantly narrower and/or shorter than uninvaded galls. Galls of social species that had not been invaded and contained adult soldiers were significantly larger than galls where soldiers were still at a larval stage, suggesting that gall size is related to gall age in these species. An hypothesis is proposed that links the timing of invasion by kleptoparasites to size of the host gall： induction of a smaller gall by host founders will reduce the period of vulnerability to invasion （before soldiers become adults） for social thrips by allowing foundresses in these smaller galls to begin laying soldierdestined eggs relatively sooner.