Electrical Penetration Graph Recording of Russian Wheat Aphid (Hemiptera: Aphididae) Feeding on Aphid-Resistant Wheat and Barley.

Biotypes of Russian wheat aphid, Diuraphis noxia (Kurdjumov), have nullified D. noxia-resistant wheat. In this study, feeding of North American D. noxia was measured in aphids fed resistant and susceptible wheat and barley using electrical penetration graph (EPG) recordings. Interactions between barley genotypes and D. noxia biotypes were significant. EPG recordings of biotype 1 aphids fed on D. noxia-resistant IBRWAGP4-7 barley plants displayed significantly more non-phloem (pathway) phase movements and significantly less sieve element phase (SEP) feeding than on susceptible plants. EPG recordings of D. noxia biotype 2 feeding are the first ever recorded, but no differences between biotype 2-susceptible and -resistant barley plants were found for any EPG parameter in biotype 2 aphids fed barley. No wheat genotype-D. noxia biotype interactions were detected, but when responses were averaged across resistant and susceptible wheat genotypes, biotype 1 displayed a significantly longer pathway phase and significantly more SEP feeding than biotype 2, and biotype 2 engaged in significantly more xylem drinking than biotype 1. IBRWAGP4-7 barley resistance to biotype 1 appears to be controlled by both intercellular factors encountered during the pathway phase and intracellular factors ingested during SEP feeding. The lack of differences in EPG parameters displayed by biotype 2 feeding on barley suggests that biotype 2 resistance in IBRWAGP4-7 barley is based on tolerance to D. noxia feeding instead of altered feeding patterns. Resistance in 'KS94H871' wheat appears to be a function of phloem, non-phloem, and xylem factors that extend the duration of pathway feeding and limit SEP feeding.

Mobilization of lipids and fortification of cell wall and cuticle are important in host defense against Hessian fly.

BACKGROUND: Wheat - Hessian fly interaction follows a typical gene-for-gene model. Hessian fly larvae die in wheat plants carrying an effective resistance gene, or thrive in susceptible plants that carry no effective resistance gene. RESULTS: Gene sets affected by Hessian fly attack in resistant plants were found to be very different from those in susceptible plants. Differential expression of gene sets was associated with differential accumulation of intermediates in defense pathways. Our results indicated that resources were rapidly mobilized in resistant plants for defense, including extensive membrane remodeling and release of lipids, sugar catabolism, and amino acid transport and degradation. These resources were likely rapidly converted into defense molecules such as oxylipins; toxic proteins including cysteine proteases, inhibitors of digestive enzymes, and lectins; phenolics; and cell wall components. However, toxicity alone does not cause immediate lethality to Hessian fly larvae. Toxic defenses might slow down Hessian fly development and therefore give plants more time for other types of defense to become effective. CONCLUSION: Our gene expression and metabolic profiling results suggested that remodeling and fortification of cell wall and cuticle by increased deposition of phenolics and enhanced cross-linking were likely to be crucial for insect mortality by depriving Hessian fly larvae of nutrients from host cells. The identification of a large number of genes that were differentially expressed at different time points during compatible and incompatible interactions also provided a foundation for further research on the molecular pathways that lead to wheat resistance and susceptibility to Hessian fly infestation.

Feeding behavior comparison of soybean aphid (Hemiptera: Aphididae) biotypes on different soybean genotypes.

Soybean aphids have become a serious pest of soybean, Glycine max L. (Merrill), since they were first detected in North America in 2000. Three soybean aphid biotypes have been documented in the United States in the last 10 yr, but few studies have been done on their feeding behavior in the United States The Electrical Penetration Graph is a convenient and successful tool to study the feeding behavior of piercing and sucking insects. This is the first attempt to study the feeding behavior differences between biotype 1 and biotype 2 on soybean genotypes using the Electrical Penetration Graph technique, and includes both resistant and susceptible soybean genotypes from Kansas and Michigan. The experiments were run for 9 h each for each genotype with a total of eight channels at a time. Results indicated that aphids feeding on susceptible genotypes had a significantly greater duration of sieve element phase than when feeding on resistant genotypes. Furthermore, the time taken to reach the first sieve element phase in resistant genotypes was significantly greater than in susceptible genotypes. Most of the aphids reached sieve element phase (> 90%) in susceptible genotypes, but only a few (< 30%) reached sieve element phase in resistant genotypes during the 9-h recording period; however, we found no differences in any other probing phases between resistant and susceptible genotypes except the number of potential drops in biotype 2. Thus, the resistance was largely associated with phloem tissues. Therefore, some biochemical, physical, or morphological factors could affect stylet penetration of aphids.

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae.

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.

Rapid mobilization of membrane lipids in wheat leaf sheaths during incompatible interactions with Hessian fly.

Hessian fly (HF) is a biotrophic insect that interacts with wheat on a gene-for-gene basis. We profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing the resistance gene H13. Our results revealed a 32 to 45% reduction in total concentrations of 129 lipid species in resistant plants during incompatible interactions within 24 h after HF attack. A smaller and delayed response was observed in susceptible plants during compatible interactions. Microarray and real-time polymerase chain reaction analyses of 168 lipid-metabolism-related transcripts revealed that the abundance of many of these transcripts increased rapidly in resistant plants after HF attack but did not change in susceptible plants. In association with the rapid mobilization of membrane lipids, the concentrations of some fatty acids and 12-oxo-phytodienoic acid (OPDA) increased specifically in resistant plants. Exogenous application of OPDA increased mortality of HF larvae significantly. Collectively, our data, along with previously published results, indicate that the lipids were mobilized through lipolysis, producing free fatty acids, which were likely further converted into oxylipins and other defense molecules. Our results suggest that rapid mobilization of membrane lipids constitutes an important step for wheat to defend against HF attack.

Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach.

The relationship between aphids and their host plants is thought to be functionally analogous to plant-pathogen interactions. Although virulence effector proteins that mediate plant defenses are well-characterized for pathogens such as bacteria, oomycetes, and nematodes, equivalent molecules in aphids and other phloem-feeders are poorly understood. A dual transcriptomic-proteomic approach was adopted to generate a catalog of candidate effector proteins from the salivary glands of the pea aphid, Acyrthosiphon pisum. Of the 1557 transcript supported and 925 mass spectrometry identified proteins, over 300 proteins were identified with secretion signals, including proteins that had previously been identified directly from the secreted saliva. Almost half of the identified proteins have no homologue outside aphids and are of unknown function. Many of the genes encoding the putative effector proteins appear to be evolving at a faster rate than homologues in other insects, and there is strong evidence that genes with multiple copies in the genome are under positive selection. Many of the candidate aphid effector proteins were previously characterized in typical phytopathogenic organisms (e.g., nematodes and fungi) and our results highlight remarkable similarities in the saliva from plant-feeding nematodes and aphids that may indicate the evolution of common solutions to the plant-parasitic lifestyle.

Testing the importance of jasmonate signalling in induction of plant defences upon cabbage aphid (Brevicoryne brassicae) attack.

BACKGROUND: Phloem-feeding aphids deprive plants of assimilates, but mostly manage to avoid causing the mechanical tissue damage inflicted by chewing insects. Nevertheless, jasmonate signalling that is induced by infestation is important in mediating resistance to phloem feeders. Aphid attack induces the jasmonic acid signalling pathway, but very little is known about the specific impact jasmonates have on the expression of genes that respond to aphid attack. RESULTS: We have evaluated the function that jasmonates have in regulating Arabidopsis thaliana responses to cabbage aphid (Brevicoryne brassicae) by conducting a large-scale transcriptional analysis of two mutants: aos, which is defective in jasmonate production, and fou2, which constitutively induces jasmonic acid biosynthesis. This analysis enabled us to determine which genes' expression patterns depend on the jasmonic acid signalling pathway. We identified more than 200 genes whose expression in non-challenged plants depended on jasmonate levels and more than 800 genes that responded differently to infestation in aos and fou2 plants than in wt. Several aphid-induced changes were compromised in the aos mutant, particularly genes connected to regulation of transcription, defence responses and redox changes. Due to jasmonate-triggered pre-activation of fou2, its transcriptional profile in non-challenged plants mimicked the induction of defence responses in wt. Additional activation of fou2 upon aphid attack was therefore limited. Insect fitness experiments revealed that the physiological consequences of fou2 mutation contributed to more effective protection against B. brassicae. However, the observed resistance of the fou2 mutant was based on antibiotic rather than feeding deterrent properties of the mutant as indicated by an analysis of aphid feeding behaviour. CONCLUSIONS: Analysis of transcriptional profiles of wt, aos and fou2 plants revealed that the expression of more than 200 genes is dependent on jasmonate status, regardless of external stimuli. Moreover, the aphid-induced response of more than 800 transcripts is regulated by jasmonate signalling. Thus, in plants lacking jasmonates many of the defence-related responses induced by infestation in wt plants are impaired. Constant up-regulation of jasmonate signalling as evident in the fou2 mutant causes reduction in aphid population growth, likely as a result of antibiotic properties of fou2 plants. However, aos mutation does not seem to affect aphid performance when the density of B. brassicae populations on plants is low and aphids are free to move around.

A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant.

In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid-plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an N-terminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.

Electrical penetration graph analysis of the feeding behavior of soybean aphids on soybean cultivars with antibiosis.

The soybean aphid, Aphis glycine Matsumura (Hemiptera: Aphididae), is a major pest of soybean. In the current study, we used the Electrical Penetration Graph technique to study feeding behavior of soybean aphids on antibiotic-resistant soybean lines KS1621, KS1613, and KS1642, and a susceptible soybean line, KS4202. We observed that soybean aphids spent significantly shorter periods of time in the sieve element phase but slightly more times in nonprobing phases in all three resistant lines than in the susceptible control. Our study suggests that resistance factors exist in the phloem of the resistant soybean lines, and that these lines may contain antixenosis in addition to antibiosis.

Hessian fly (Mayetiola destructor) attack causes a dramatic shift in carbon and nitrogen metabolism in wheat.

Carbon and nitrogen (C/N) metabolism and allocation within the plant have important implications for plant-parasite interactions. Many plant parasites manipulate the host by inducing C/N changes that benefit their own survival and growth. Plant resistance can prevent this parasite manipulation. We used the wheat-Hessian fly (Mayetiola destructor) system to analyze C/N changes in plants during compatible and incompatible interactions. The Hessian fly is an insect but shares many features with plant pathogens, being sessile during feeding stages and having avirulence (Avr) genes that match plant resistance genes in gene-for-gene relationships. Many wheat genes involved in C/N metabolism were differentially regulated in plants during compatible and incompatible interactions. In plants during compatible interactions, the content of free carbon-containing compounds decreased 36%, whereas the content of free nitrogen-containing compounds increased 46%. This C/N shift was likely achieved through a coordinated regulation of genes in a number of central metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and amino-acid synthesis. Our data on plants during compatible interactions support recent findings that Hessian fly larvae create nutritive cells at feeding (attack) sites and manipulate host plants to enhance their own survival and growth. In plants during incompatible interactions, most of the metabolic genes examined were not affected or down-regulated.

Chlorophyll loss caused by soybean aphid (Hemiptera: Aphididae) feeding on soybean.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a worldwide pest of soybean, Glycine max (L.) Merr. Studies to find control methods were initiated in 2000 when it was first detected in North America. A. glycines can reduce yields by as much as 50%, and it is the vector of several viral diseases. A. glycines removes phloem sap, which can result in a reduction of chlorophyll content. Quantification of chlorophyll loss caused by A. glycines feeding on soybean is of vital importance. The SPAD-502 chlorophyll meter is a device that has been used to measure chlorophyll loss caused by nonchewing insects. Chlorophyll loss was studied in no-choice tests on the infested and uninfested leaves of a susceptible check (KS4202). The minimum combined number of days and aphids needed to detect significant chlorophyll loss was 30 aphids confined for 10 d. In a similar experiment, seven resistant entries and two susceptible checks were evaluated. There was no significant chlorophyll reduction between infested and uninfested leaves of five of the resistant entries (K1621, K1639, Pioneer 95B97, Dowling, and Jackson). Percentage of loss of chlorophyll in the susceptible checks was approximately 40%; Jackson and Dowling had a significantly lower percentage loss (13 and 16%, respectively) compared with the susceptible checks. The percentages of chlorophyll loss of K1621, K1639, and Pioneer 95B97 were not statistically different from the percentage of loss of Jackson.

Feeding behavior by the soybean aphid (Hemiptera: Aphididae) on resistant and susceptible soybean genotypes.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a major pest of soybean, Glycine max (L.) Merr. Since 2000, when A. glycines was detected in the United States, several studies on this insect have been done in different areas, but there is no report of any studies of stylet penetration behavior by A. glycines on resistant and susceptible soybean. Assessment of feeding behavior of this aphid species was compared on four resistant entries (K1639, Pioneer 95B97, Dowling, and Jackson) and a susceptible check (KS4202) by using the electrical penetration graph (EPG) technique. Feeding behavior of A. glycines adults was recorded during a 9-h period. The average time needed to reach the first sieve element phase by A. glycines was 3.5 h in KS4202, whereas it was 7.5 h in the resistant entries. The total duration in the sieve element phase was longer than an hour in KS4202, and only 2 to 7 min in the resistant entries. These results suggest that morphological or chemical factors in the phloem tissue of resistant plants affect stylet penetration activities of A. glycines. In the majority of the recordings, however, the aphid stylet reached the xylem phase before penetrating the sieve element, and the time that aphids spent ingesting xylem sap was not different among all entries. Therefore, it is possible that xylem sap in the resistant entries may contain toxic substances that change aphid behavior and that affect further activities in the sieve element phase.

Characterization of antibiosis and antixenosis to the soybean aphid (Hemiptera: Aphididae) in several soybean genotypes.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is an introduced pest of soybean, Glycine max (L.) Merr., in North America, and it can reduce yields by 50%. Since 2000, when A. glycines was first detected in the United States, studies of this insect and possible control methods have been initiated. Plant resistance to this aphid species is one important component of integrated control. Reproduction of A. glycines was compared on 240 soybean entries in a pesticide-free greenhouse. Eleven entries had fewer nymphs produced, compared with the susceptible checks, and these entries were used in follow-up experiments to assess antibiosis and antixenosis. Antibiosis was estimated in true no-choice tests, in which adults were confined individually in double-sided sticky cages stuck to the upper side of leaves. Antixenosis was assessed in choice tests, in which all entries were planted in a single pot. Adult aphids were placed in the center of the pot, and 24 h later the number of adults on each plant was counted. Of the 11 entries evaluated, nine showed a moderate antibiotic effect to A. glycines, and the other two entries (K1639 and Pioneer 95B97) showed not only a strong antibiotic effect but also exhibited antixenosis as a category of resistance to A. glycines. The resistant soybean entries found in this work are potential sources for A. glycines control.

RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum.

Abstract Injection of siRNA (small interfering RNA) into parthenogenetic adult pea aphids (Acyrthosiphon pisum) is shown here to lead to depletion of a target salivary gland transcript. The siRNA was generated from double stranded RNA that covered most of the open reading frame of the transcript, which we have called Coo2. The Coo2 transcript level decreases dramatically over a 3-day period after injection of siRNA. With a lag of 1 to 2 days, the siCoo2-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections into adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands and suggests that siCoo2-RNA injections can be a useful positive control in such studies.

Tritrophic interaction of parasitoid Lysiphlebus testaceipes (Hymenoptera: Aphidiidae), greenbug, Schizaphis graminum (Homoptera: Aphididae), and greenbug-resistant sorghum hybrids.

Interactions of the parasitoid Lysiphlebus testaceipes (Cresson) and the greenbug, Schizaphis graminum (Rondani), on greenbug-resistant 'Cargill 607E' (antibiosis), 'Cargill 797' (primarily tolerance), and -susceptible 'Golden Harvest 510B' sorghum, Sorghum bicolor (L.) Moench, were tested using three levels of biotype I greenbug infestation. The parasitoid infestation rate was 0.5 female and 1.0 male L. testaceipes per plant. For all three greenbug infestation levels, the parasitoid brought the greenbug under control (i.e., prevented the greenbugs from killing the plants) on both resistant hybrids, but it did not prevent heavy leaf damage at the higher greenbug infestation rates. At the low greenbug infestation rate (50 greenbugs per resistant plant when parasitoids were introduced), greenbugs damaged 5 and 18% of the total leaf area on 'Cargill 797' and 'Cargill 607E', respectively, before greenbugs were eliminated. Leaf damage was higher for the intermediate infestation study (120 greenbugs per plant), 21% and 30% leaf area were damaged on the resistant sorghum hybrids 'Cargill 797' and 'Cargill 607E', respectively. At the high greenbug infestation rate (300 greenbugs per plant), heavy damage occurred: 61% on 'Cargill 607E' and 75% on 'Cargill 797'. The parasitoids did not control greenbugs on the susceptible sorghum hybrid 'Golden Harvest 510B'. L. testaceipes provided comparable control on both greenbug-resistant hybrids. This study supports previous studies indicating that L. testaceipes is effective in controlling greenbugs on sorghum with antibiosis resistance to greenbugs. Furthermore, new information is provided indicating that L. testaceipes is also effective in controlling greenbugs on a greenbug-tolerant hybrid.

Molecular mapping of sorghum genes expressing tolerance to damage by greenbug (Homoptera: Aphididae).

Genetic linkage maps are fundamental for the localization of genes conferring tolerance to greenbug, Schizaphis graminum (Rondani), feeding damage in sorghum, Sorghum bicolor (L.) Moench. Thirteen linkage groups (LGs) containing 60 simple sequence repeat (SSR) loci were mapped by using a set of sorghum recombinant inbred lines (RILs) obtained from the cross '96-4121' (greenbug-tolerant parent) x Redlan (greenbug-susceptible parent). The LG spanned a distance of 603.5 cM, with the number of loci per LG varying from 2 to 14. Seventeen additional SSR loci were unlinked at a log of odds value of 3.0. Based on chlorophyll loss occurring after greenbug feeding, visual damage ratings, and soil plant analysis development (SPAD), chlorophyll-loss indices were recorded for each RIL and for the parents used in the cross. Composite-interval mapping identified three quantitative trait loci (QTLs) associated with biotype I and five QTLs associated with biotype K. The amount of phenotypic variation explained by these QTLs ranged from 9 to 19.6%. The identification of QTLs that influence greenbug tolerance will not only facilitate the use of marker-assisted selection in sorghum breeding programs but also will provide a solid foundation for detailed characterization of individual loci implicated in greenbug tolerance in sorghum.

Pectinmethylesterase from the rice weevil, Sitophilus oryzae: cDNA isolation and sequencing, genetic origin, and expression of the recombinant enzyme.

A cDNA clone encoding pectinmethylesterase of the rice weevil, Sitophilus oryzae (L.) has been isolated and sequenced. The cDNA clone was expressed in cultured insect cells and active pectinmethylesterase was purified from the culture medium, thus confirming that the cDNA encodes pectinmethylesterase. In situ hybridization indicated that the enzyme's transcript was present in the midgut. Weevils treated with tetracycline so that they lack genes of known symbiotic organisms still contained the pectinmethylesterase gene, indicating that the gene is encoded by the rice weevil genome. The rice weevil enzyme is most similar in sequence to bacterial pectinmethylesterases. Given this and the enzyme's apparently rather general absence from animal species, we suggest the possibility that this gene was transferred horizontally to an ancient weevil, possibly from a bacterial symbiont, and exists in Sitophilus species now as a result of that ancestral horizontal transfer.

Polygalacturonase from Sitophilus oryzae: possible horizontal transfer of a pectinase gene from fungi to weevils.

Endo-polygalacturonase, one of the group of enzymes known collectively as pectinases, is widely distributed in bacteria, plants and fungi. The enzyme has also been found in several weevil species and a few other insects, such as aphids, but not in Drosophila melanogaster, Anopheles gambiae, or Caenorhabditis elegans or, as far as is known, in any more primitive animal species. What, then, is the genetic origin of the polygalacturonases in weevils? Since some weevil species harbor symbiotic microorganisms, it has been suggested, reasonably, that the symbionts' genomes of both aphids and weevils, rather than the insects' genomes, could encode polygalacturonase. We report here the cloning of a cDNA that encodes endo-polygalacturonase in the rice weevil, Sitophilus oryzae (L.), and investigations based on the cloned cDNA. Our results, which include analysis of genes in antibiotic-treated rice weevils, indicate that the enzyme is, in fact, encoded by the insect genome. Given the apparent absence of the gene in much of the rest of the animal kingdom, it is therefore likely that the rice weevil polygalacturonase gene was incorporated into the weevil's genome by horizontal transfer, possibly from a fungus.

Pyrosequencing reveals the predominance of pseudomonadaceae in gut microbiome of a gall midge.

Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs) were obtained at the >97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall midge, and provides a foundation for future studies to elucidate the roles of gut microbes in Hessian fly virulence and biology.

The structure of rice weevil pectin methylesterase.

Rice weevils (Sitophilus oryzae) use a pectin methylesterase (EC 3.1.1.11), along with other enzymes, to digest cell walls in cereal grains. The enzyme is a right-handed ß-helix protein, but is circularly permuted relative to plant and bacterial pectin methylesterases, as shown by the crystal structure determination reported here. This is the first structure of an animal pectin methylesterase. Diffraction data were collected to 1.8 Šresolution some time ago for this crystal form, but structure solution required the use of molecular-replacement techniques that have been developed and similar structures that have been deposited in the last 15 years. Comparison of the structure of the rice weevil pectin methylesterase with that from Dickeya dandantii (formerly Erwinia chrysanthemi) indicates that the reaction mechanisms are the same for the insect, plant and bacterial pectin methylesterases. The similarity of the structure of the rice weevil enzyme to the Escherichia coli lipoprotein YbhC suggests that the evolutionary origin of the rice weevil enzyme was a bacterial lipoprotein, the gene for which was transferred to a primitive ancestor of modern weevils and other Curculionidae. Structural comparison of the rice weevil pectin methylesterase with plant and bacterial enzymes demonstrates that the rice weevil protein is circularly permuted relative to the plant and bacterial molecules.