A mixed diet of toxic plants enables increased feeding and anti-predator defense by an insect herbivore.

Some insect herbivores sequester plant secondary metabolites (PSMs) for their own defense, raising the interesting possibility that grazing herbivores are defended by combinations of PSMs from different plant species. In this study, we tested the hypothesis that the grazing caterpillar, Grammia incorrupta, deters the ant, Aphaenogaster cockerelli, by eating a mixture of plants containing iridoid glycosides (IGs) and those containing pyrrolizidine alkaloids (PAs), and that this deterrence is greater than that attained by eating either plant alone. This hypothesis was tested against the non-mutually exclusive hypothesis that mixing plants containing PAs with those containing IGs improves growth performance. Caterpillar survival and growth were measured on three experimental diets: a PA plant, an IG plant, and a mixture of the two. We measured the degree of deterrence associated with these, and an additional experimental diet devoid of PSMs at naturally occurring A. cockerelli nests. Caterpillars fed both plants gained more mass than those fed either plant alone, but took longer to develop. These differences were not caused by diet-based variation in growth efficiency, but by eating more food when offered the mixed-plant diet relative to single-plant diets. The mixed diet was shown to provide deterrence to ants, whereas caterpillars fed single-plant diets were not significantly more deterrent than caterpillars that had eaten the PSM-free diet. We hypothesize that enhanced defense results from increased food consumption in response to multiple plant species, perhaps leading to greater PSM sequestration. Through this mechanism, bottom-up and top-down effects may mutually reinforce the grazing dietary strategy.

Endosulfatases SULF1 and SULF2 limit Chlamydia muridarum infection.

The first step in attachment of Chlamydia to host cells is thought to involve reversible binding to host heparan sulfate proteoglycans (HSPGs), polymers of variably sulfated repeating disaccharide units coupled to diverse protein backbones. However, the key determinants of HSPG structure that are involved in Chlamydia binding are incompletely defined. A previous genome-wide Drosophila RNAi screen suggested that the level of HSPG 6-O sulfation rather than the identity of the proteoglycan backbone maybe a critical determinant for binding. Here, we tested in mammalian cells whether SULF1 or SULF2, human endosulfatases, which remove 6-O sulfates from HSPGs, modulate Chlamydia infection. Ectopic expression of SULF1 or SULF2 in HeLa cells, which decreases cell surface HSPG sulfation, diminished C. muridarum binding and decreased vacuole formation. ShRNA depletion of endogenous SULF2 in a cell line that primarily expresses SULF2 augmented binding and increased vacuole formation. C. muridarum infection of diverse cell lines resulted indownregulation of SULF2 mRNA. In a murine model of acute pneumonia, mice genetically deficient in both endosulfatases or in SULF2 alone demonstrated increased susceptibility to C. muridarum lung infection. Collectively, these studies demonstrate that the level of HSPG 6-O sulfation is a critical determinant of C. muridarum infection in vivo and that 6-O endosulfatases are previously unappreciated modulators of microbial pathogenesis.

Revisiting the evolution of ecological specialization, with emphasis on insect-plant interactions.

Ecological specialization is a fundamental and well-studied concept, yet its great reach and complexity limit current understanding in important ways. More than 20 years after the publication of D. J. Futuyma and G. Moreno's oft-cited, major review of the topic, we synthesize new developments in the evolution of ecological specialization. Using insect-plant interactions as a model, we focus on important developments in four critical areas: genetic architecture, behavior, interaction complexity, and macroevolution. We find that theory based on simple genetic trade-offs in host use is being replaced by more subtle and complex pictures of genetic architecture, and multitrophic interactions have risen as a necessary framework for understanding specialization. A wealth of phylogenetic data has made possible a more detailed consideration of the macroevolutionary dimension of specialization, revealing (among other things) bidirectionality in transitions between generalist and specialist lineages. Technological advances, including genomic sequencing and analytical techniques at the community level, raise the possibility that the next decade will see research on specialization spanning multiple levels of biological organization in non-model organisms, from genes to populations to networks of interactions in natural communities. Finally, we offer a set of research questions that we find to be particularly pressing and fruitful for future research on ecological specialization.

Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis.

Heparan sulfate (HS) proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small-cell lung carcinomas (NSCLCs). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2(+) NSCLC cell lines, including two, which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer.

Host specificity of Lepidoptera in tropical and temperate forests.

For numerous taxa, species richness is much higher in tropical than in temperate zone habitats. A major challenge in community ecology and evolutionary biogeography is to reveal the mechanisms underlying these differences. For herbivorous insects, one such mechanism leading to an increased number of species in a given locale could be increased ecological specialization, resulting in a greater proportion of insect species occupying narrow niches within a community. We tested this hypothesis by comparing host specialization in larval Lepidoptera (moths and butterflies) at eight different New World forest sites ranging in latitude from 15 degrees S to 55 degrees N. Here we show that larval diets of tropical Lepidoptera are more specialized than those of their temperate forest counterparts: tropical species on average feed on fewer plant species, genera and families than do temperate caterpillars. This result holds true whether calculated per lepidopteran family or for a caterpillar assemblage as a whole. As a result, there is greater turnover in caterpillar species composition (greater beta diversity) between tree species in tropical faunas than in temperate faunas. We suggest that greater specialization in tropical faunas is the result of differences in trophic interactions; for example, there are more distinct plant secondary chemical profiles from one tree species to the next in tropical forests than in temperate forests as well as more diverse and chronic pressures from natural enemy communities.

Climatic unpredictability and parasitism of caterpillars: implications of global warming.

Insect outbreaks are expected to increase in frequency and intensity with projected changes in global climate through direct effects of climate change on insect populations and through disruption of community interactions. Although there is much concern about mean changes in global climate, the impact of climatic variability itself on species interactions has been little explored. Here, we compare caterpillar-parasitoid interactions across a broad gradient of climatic variability and find that the combined data in 15 geographically dispersed databases show a decrease in levels of parasitism as climatic variability increases. The dominant contribution to this pattern by relatively specialized parasitoid wasps suggests that climatic variability impairs the ability of parasitoids to track host populations. Given the important role of parasitoids in regulating insect herbivore populations in natural and managed systems, we predict an increase in the frequency and intensity of herbivore outbreaks through a disruption of enemy-herbivore dynamics as climates become more variable.

Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura.

The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxified by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea. Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as "drug sources". However, unlike E. acrea, G. geneura is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-esterified yielding two distinct classes of insect-specific ester alkaloids, the creatonotines, also present in E. acrea, and the callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterification. Open-chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines and callimorphines by trans-esterification in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is presented that during this processing the necine base (trachelanthamidine) is converted into its 7-(R)-hydroxy derivative (turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted. The possible function of insect-specific trans-esterification in the acquisition of necine bases derived from plant acquired alkaloids, especially from those that cannot be maintained through all life-stages, is discussed.

Specific recognition, detoxification and metabolism of pyrrolizidine alkaloids by the polyphagous arctiid Estigmene acrea.

Evidence is presented that the polyphagous arctiid Estigmene acrea is well adapted to sequester and specifically handle pyrrolizidine alkaloids of almost all known structural types representative of the major plant families with pyrrolizidine alkaloid-containing species, i.e. Asteraceae with the tribes Senecioneae and Eupatorieae, Boraginaceae, Fabaceae, Apocynaceae and Orchidaceae. The adaptation of E. acrea to pyrrolizidine alkaloids includes a number of specialized characters: (i) highly sensitive recognition of alkaloid sources by pyrrolizidine alkaloid-specific taste receptors; (ii) detoxification of pyrrolizidine alkaloids by N-oxidation catalyzed by a specific flavin-dependent monooxygenase; (iii) transfer and maintenance of all types of pyrrolizidine N-oxides through all developmental stages; (iv) conversion of the various structures into the male courtship pheromone hydroxydanaidal most probably through retronecine and insect specific retronecine esters (creatonotines) as common intermediates; (v) specific integration into mating behavior and defense strategies. Toxic otonecine derivatives, e.g. the senecionine analogue senkirkine, which often accompany the common retronecine derivatives and which cannot be detoxified by N-oxidation do not affect the development of E. acrea larvae. Senkirkine is not sequestered at all. Non-toxic 1,2-saturated platynecine derivatives that frequently occur together with toxic retronecine esters are sequestered and metabolized to hydroxydanaidal, indicating the ability of E. acrea to aromatize saturated pyrrolizidines. Although pyrrolizidine alkaloids, even if they are offered continuously at a high level (2%) in the larval diet, are non-toxic, E. acrea larvae are not able to develop exclusively on a pyrrolizidine alkaloid-containing plant like Crotalaria. Therefore, E. acrea appears to be specifically adapted to exploit pyrrolizidine alkaloid-containing plants as "drug source" but not as a food source.

Acquired and partially de novo synthesized pyrrolizidine alkaloids in two polyphagous arctiids and the alkaloid profiles of their larval food-plants.

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.

Changes in taste receptor cell sensitivity in a polyphagous caterpillar reflect carbohydrate but not protein imbalance.

Caterpillars of the polyphagous arctiid, Grammia geneura, have a single cell in the medial galeal sensillum that responds to some sugars and to some amino acids. After conditioning on artificial diet containing unbalanced amounts of carbohydrate and protein, the responses of this cell alter. After protein-biased food it increases slightly, but after carbohydrate-biased food it decreases. Responses to both sucrose and amino acids change in the same direction and the changes would not provide the information necessary to redress a shortage of protein. The lateral galeal sensillum contains one cell that responds to fructose and another responding to some amino acids. The responses of each of these cells in the lateral sensillum are not consistently affected by conditioning diets. After conditioning for 20 h on a protein- or carbohydrate-biased diet, the insects started to feed without delay if offered carbohydrate-biased diet, but only after a pause if given protein-biased diet. This occurred irrespective of the conditioning diet. The duration of the first feeding bout was also longer on carbohydrate-biased diet and the longest bouts followed protein-biased conditioning.

Loss of gustatory responses to pyrrolizidine alkaloids after their extensive ingestion in the polyphagous caterpillar Estigmene acrea.

Electrophysiological recordings from taste sensilla of the caterpillar Estigmene acrea with the pyrrolizidine alkaloid (PA) seneciphylline N-oxide demonstrated that extensive feeding on plants rich in PAs caused a loss in response of the PA-sensitive cell in the lateral styloconic sensillum on the galea. The effect could be repeated using pure PAs fed to the insect in synthetic diets and by injection of PA into the hemolymph. The sensitivity loss lasted for approximately two hours and was less pronounced in individuals that had been reared on PA-containing food. Behavioral experiments and field observations demonstrate a parallel reduction in responsiveness to PAs and to PA-containing plants.

Experience influences gustatory responsiveness to pyrrolizidine alkaloids in the polyphagous caterpillar, Estigmene acrea.

Electrophysiological recordings from taste sensilla of the caterpillar Estigmene acrea with the pyrrolizidine alkaloid, seneciphylline N-oxide, demonstrated that prior feeding on plants with pyrrolizidine alkaloids caused an increase in responsiveness of the PA-sensitive cells in two sensilla, relative to feeding on plants without such chemicals. Rearing on synthetic diet without pyrrolizidine alkaloids for up to seven generations caused a continuous decline in responsiveness, that could be reversed by experience with powdered Crotalaria pumila in the diet or by pure pyrrolizidine alkaloid, monocrotaline, in the diet. Response to the cardiac glycoside, ouabain, that stimulates one of the two pyrrolizidine alkaloid-sensitive cells, showed a similar decline. Pyrrolizidine alkaloids had no measurable effect on growth and development. Responses in all other taste cells were unaffected. The data are discussed in relation to the possible adaptive significance and the possible mechanisms involved.

The olfactory receptor family album.

Analysis of the human genome draft sequences has revealed a more complete portrait of the olfactory receptor gene repertoire in humans than was available previously. The new information provides a basis for deeper analysis of the functions of the receptors, and promises new insights into the evolutionary history of the family.

Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes.

Lymphocytes home to lymph nodes, using L-selectin to bind specific ligands on high endothelial venules (HEV). In vitro studies implicate GlcNAc-6-sulfate as an essential posttranslational modification for ligand activity. Here, we show that genetic deletion of HEC-GlcNAc6ST, a sulfotransferase that is highly restricted to HEV, results in the loss of the binding of recombinant L-selectin to the luminal aspect of HEV, elimination of lymphocyte binding in vitro, and markedly reduced in vivo homing. Reactivity with MECA 79, an adhesion-blocking mAb that stains HEV in lymph nodes and vessels in chronic inflammatory sites, is also lost from the luminal aspects of HEV. These results establish a critical role for HEC-GlcNAc6ST in lymphocyte trafficking and suggest it as an important therapeutic target.

Contrasting responses to desiccation and starvation by eggs and neonates of two lepidoptera.

We examined the effects of desiccation on eggs and first-instar larvae of two species of Lepidoptera, Grammia geneura (Arctiidae) and Manduca sexta (Sphingidae). Grammia geneura occurs primarily in grasslands and savannas of the southwestern United States; M. sexta co-occurs with G. geneura but also is cosmopolitan across much of the Western Hemisphere. Eggs of G. geneura exposed to 0% relative humidity (RH) lost water much less rapidly (7.6 microg d(-1); 2.4% d(-1)) than did eggs of M. sexta (79.5 microg d(-1); 5.7% d(-1)). Eggs of both species survived at rates exceeding 75% at both 0% and 85% RH. Neonates of the two species responded differently to desiccation and starvation. In 85% RH, larval G. geneura survived at high rates (>80%) without access to food or water up to day 17, and in 0% RH, they survived at rates exceeding 50% through the first 10 d. Larvae at 0% RH lost mass very slowly (7.2 microg d(-1); 2.9% d(-1)), which was attributable both to low rates of water loss and to an ability to reduce metabolic rate to low levels. Larval M. sexta, in contrast, had rates of mortality that were much higher: after 1 d, fewer than 30% were alive in either group, and by about 1.5 d, all were dead. Neonate M. sexta also lost mass much more rapidly at 0% RH, about 329 microg d(-1). Water from metabolism appeared to contribute significantly to the water budget of G. geneura but not of M. sexta. These data show that G. geneura and M. sexta can inhabit similar macroclimates via remarkably different physiologies.

The function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku.

The telomerase enzyme lengthens telomeres, an activity essential for chromosome stability in most eukaryotes. The enzyme is composed of a specialized reverse transcriptase and a template RNA. In Saccharomyces cerevisiae, overexpression of TLC1, the telomerase RNA gene, disrupts telomeric structure. The result is both shortened telomere length and loss of a special chromatin structure that normally silences telomere-proximal genes. Because telomerase function is not required for telomeric silencing, we postulated that the dominant-negative effect caused by overexpression of TLC1 RNA originates in a normal interaction between the RNA and an unknown telomeric factor important for silencing; the overexpressed RNA presumably continues to bind the factor and compromises its function. Here we show that a 48-nt stem-loop structure within the 1.3-kb TLC1 RNA is necessary and sufficient for disrupting telomeric silencing and shortening telomeres. Moreover, this short RNA sequence appears to function through an interaction with the conserved DNA end-binding protein Ku. We propose that, in addition to its roles in telomeric silencing, homologous recombination and non-homologous end-joining (NHEJ), S. cerevisiae Ku also helps to recruit or activate telomerase at the telomere through an interaction with this stem-loop of TLC1 RNA.

Molecular mechanisms underlying differential odor responses of a mouse olfactory receptor.

The prevailing paradigm for G protein-coupled receptors is that each receptor is narrowly tuned to its ligand and closely related agonists. An outstanding problem is whether this paradigm applies to olfactory receptor (ORs), which is the largest gene family in the genome, in which each of 1,000 different G protein-coupled receptors is believed to interact with a range of different odor molecules from the many thousands that comprise "odor space." Insights into how these interactions occur are essential for understanding the sense of smell. Key questions are: (i) Is there a binding pocket? (ii) Which amino acid residues in the binding pocket contribute to peak affinities? (iii) How do affinities change with changes in agonist structure? To approach these questions, we have combined single-cell PCR results [Malnic, B., Hirono, J., Sato, T. & Buck, L. B. (1999) Cell 96, 713-723] and well-established molecular dynamics methods to model the structure of a specific OR (OR S25) and its interactions with 24 odor compounds. This receptor structure not only points to a likely odor-binding site but also independently predicts the two compounds that experimentally best activate OR S25. The results provide a mechanistic model for olfactory transduction at the molecular level and show how the basic G protein-coupled receptor template is adapted for encoding the enormous odor space. This combined approach can significantly enhance the identification of ligands for the many members of the OR family and also may shed light on other protein families that exhibit broad specificities, such as chemokine receptors and P450 oxidases.

Analysis of the molecular basis for octanal interactions in the expressed rat 17 olfactory receptor.

Expression studies have shown that the rat I7 olfactory receptor (OR-I7) responds preferentially to the aldehyde n-octanal. We wished to predict which residues in OR-I7 bind octanal and how the biophysical properties of these residues determine the receptor's odor selectivity. Building on our previous work on aldehyde interactions in olfactory receptors, we constructed a molecular model of OR-I7 based on the 7.5 A resolution three-dimensional map of rhodopsin. Octanal was automatically docked in the model. The results predicted an odor-binding pocket approximately 10 A from the extracellular surface, in a location similar to the epinephrine-binding pocket of the beta-adrenergic receptor and the odor-binding pocket of a previous olfactory receptor model. A lysine on TM4 and an aspartate on TM5 interacted with the aldehyde moiety of octanal. Hydrophobic residues formed Van der Waals contacts with the hydrocarbon portion of octanal. We docked related odor compounds and found that the predicted affinities compared favorably with experimental results. We also tested a number of amino acid substitutions in order to predict their effects on octanal affinity and provide leads for future experimental work.

Sensitivity to chemically diverse phagostimulants in a single gustatory neuron of a polyphagous caterpillar.

Caterpillars of the arctiid moth, Grammia geneura, are polyphagous, but species of Plantago are amongst their preferred food plants. A neuron in the medial styloconic sensillum on the galea has been shown to have a general phagostimulatory function. Experiments with binary mixtures and cross-adaptation have demonstrated that it responds to some sugars, to several amino acids, and also to catalpol. Catalpol is a plant secondary compound in Plantago and a phagostimulant for the caterpillars. The possible significance of combining sensitivity to nutrient compounds with sensitivity to a secondary compound is discussed.

Olfactory Receptor Database: a database of the largest eukaryotic gene family.

The Olfactory Receptor Database (ORDB) is a WWW-accessible database that stores data on Olfactory Receptor-like molecules (ORs) and has been open to the public since June 1996. It contains a public and a private area. The public area includes published DNA and protein sequence data for ORs, links to OR models and data on their expression, chromosomal localization and source organism, as well as (i) links to bibliography through PubMed and (ii) interactive WWW-based tools, such as BLAST homology searching. The private area functions as a service to laboratories that are actively cloning receptors. Source laboratories enter the sequences of the receptor clones they have characterized to the private database and can search for identical or near identical OR sequences in both public and private databases. If another laboratory has cloned and deposited an identical or closely matching sequence there are means for communication between the laboratories to help avoid duplication of work. ORDB is available via the WWW at http://crepe.med.yale.edu/ORDB/HTML