An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the ß-keto acyl-CoA side chain of an ascaroside intermediate during ß-oxidation, leading to decarboxylation and formation of the MK. Using comparative metabolomics, we identify additional ACOT-15-dependent metabolites, including an unusual piperidyl-modified ascaroside, reminiscent of the alkaloid pelletierine. The ß-keto acid generated by ACOT-15 likely couples to 1-piperideine to produce the piperidyl ascaroside, which is much less dauer-inducing than the dauer pheromone, asc-C6-MK (ascr#2, 1). The bacterial food provided influences production of the piperidyl ascaroside by the worm. Our work shows how the biosynthesis of MK- and piperidyl ascarosides intersect and how bacterial food may impact chemical signaling in the worm.

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

Reinvestigation of sex pheromone biosynthesis in the moth Trichoplusiani reveals novel quantitative control mechanisms.

Many species of moths have a common control mechanism for synthesizing sex pheromone: the circadian release of pheromone biosynthesis-activation neuropeptide (PBAN) that switches pheromone synthesis on/off during the day. One apparent exception to this is the noctuid moth Trichoplusia ni (Hübner), in which pheromone synthesis appears continuous through the photoperiod, with circadian release of PBAN controlling emission rate of pheromone during calling. Sex pheromone biosynthesis was reinvestigated in T. ni using stable isotope tracer-tracee and gland sampling techniques to ascertain how pheromone quantities in gland cells and on the gland cuticular surface varied and were controlled. It was found that (i) carbohydrate from adult female feeding is used to synthesize sex pheromone, (ii) most of the stored acetate ester pheromone component(s) is contained in gland cells, (iii) a large pool of pheromone is synthesized and stored through the photoperiod with a slow turnover rate, (iv) although pheromone is synthesized throughout the photoperiod, its rate can vary, influenced by release of PBAN and possibly by an unidentified head factor, with both affecting carbohydrate uptake into the acetyl CoA pheromone precursor pool, and (v) as suggested previously, PBAN also influences translocation of pheromone out of the cell to the cuticular surface, possibly by causing breakdown of intracellular lipid droplets storing pheromone molecules. This work suggests that the quantitative synthesis and emission of pheromone in T. ni, and possibly other moths, is regulated by multiple complementary biochemical mechanisms.

Metabolomics and transcriptomics of pheromone biosynthesis in an aggressive forest pest Ips typographus.

Eurasian spruce bark beetle, Ips typographus, is a destructive pest in spruce forests. The ability of I. typographus to colonise host trees depends on its massive aggregation behaviour mediated by aggregation pheromones, consisting of 2-methyl-3-buten-2-ol and cis-verbenol. Other biologically active compounds such as ipsdienol and verbenone have also been detected in the beetle. Biosynthesis of 2-methyl-3-buten-2-ol and ipsdienol de novo from mevalonate and that of cis-verbenol from α-pinene sequestrated from the host have been reported in preliminary studies. However, knowledge on the molecular mechanisms underlying pheromone biosynthesis in this pest is currently limited. In this study, we performed metabolomic and differential gene expression (DGE) analysis for the pheromone-producing life stages of I. typographus. The highest amounts of 2-methyl-3-buten-2-ol (238 ng/gut) and cis-verbenol (23 ng/gut) were found in the fed male gut (colonisation stage) and the immature male gut (early stage), respectively. We also determined the amount of verbenyl oleate (the possible storage form of cis-verbenol), a monoterpenyl fatty acid ester, to be approximately 1604 ng/mg in the immature stage in the beetle body. DGE analysis revealed possible candidate genes involved in the biosynthesis of the quantified pheromones and related compounds. A novel hemiterpene-synthesising candidate isoprenyl-di-phosphate synthase Ityp09271 gene proposed for 2-methyl-3-buten-2-ol synthesis was found to be highly expressed only in the fed male beetle gut. Putative cytochrome P450 genes involved in cis/trans-verbenol synthesis and an esterase gene Ityp11977, which could regulate verbenyl oleate synthesis, were identified in the immature male gut. Our findings from the molecular analysis of pheromone-producing gene families are the first such results reported for I. typographus. With further characterisation of the identified genes, we can develop novel strategies to disrupt the aggregation behaviour of I. typographus and thereby prevent vegetation loss.

Function of mevalonate pathway genes in the synthesis of frontalin in Chinese white pine beetle, Dendroctonus armandi (curculionidae: Scolytinae).

The Chinese white pine beetle (Dendroctonus armandi Tsai and Li) is a significant pest of pine forests in the Qinling and Bashan Mountains of China. Adult males commonly produce frontalin using precursors synthesized through the mevalonate pathway, which is regulated by juvenile hormone III (JHIII). In this study, the expression levels of mevalonate pathway genes were quantified after phloem feeding and topical application of the JHIII solution. The frontalin was quantified by gas chromatography-mass spectrometry. Both the phloem feeding and JHIII treatments produced an evident upregulation in the male gut, mainly in 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). Moreover, HMGS, HMGR, isopentenyl diphosphate isomerase, and geranyl diphosphate synthase/farnesyl diphosphate synthase were upregulated in fed and JHIII-stimulated males of D. armandi under both conditions (solitary and paired). The expression levels were higher in paired compared to solitary males. Males had higher expression levels compared with females. Correspondingly, the phloem-feeding males produced more frontalin than JHIII-treated males, and the production of frontalin was higher in paired males than in solitary males. The knockdown of mevalonate pathway genes using RNAi in vivo effectively reduced the messenger RNA level of these genes and inhibited the production of frontalin. Among them, the silencing of HMGR or HMGS genes reduced the synthesis of frontalin most significantly.

Evolutionary history of mental glands in turtles reveals a single origin in an aquatic ancestor and recurrent losses independent of macrohabitat.

Despite the relevance of chemical communication in vertebrates, comparative examinations of macroevolutionary trends in chemical signaling systems are scarce. Many turtle and tortoise species are reliant on chemical signals to communicate in aquatic and terrestrial macrohabitats, and many of these species possess specialized integumentary organs, termed mental glands (MGs), involved in the production of chemosignals. We inferred the evolutionary history of MGs and tested the impact of macrohabitat on their evolution. Inference of ancestral states along a time-calibrated phylogeny revealed a single origin in the ancestor of the subclade Testudinoidea. Thus, MGs represent homologous structures in all descending lineages. We also inferred multiple independent losses of MGs in both terrestrial and aquatic clades. Although MGs first appeared in an aquatic turtle (the testudinoid ancestor), macrohabitat seems to have had little effect on MG presence or absence in descendants. Instead, we find clade-specific evolutionary trends, with some clades showing increased gland size and morphological complexity, whereas others exhibiting reduction or MG loss. In sister clades inhabiting similar ecological niches, contrasting patterns (loss vs. maintenance) may occur. We conclude that the multiple losses of MGs in turtle clades have not been influenced by macrohabitat and that other factors have affected MG evolution.

Allelopathic effect of Artemisia argyi on the germination and growth of various weeds.

Allelopathy means that one plant produces chemical substances to affect the growth and development of other plants. Usually, allelochemicals can stimulate or inhibit the germination and growth of plants, which have been considered as potential strategy for drug development of environmentally friendly biological herbicides. Obviously, the discovery of plant materials with extensive sources, low cost and markedly allelopathic effect will have far-reaching ecological impacts as the biological herbicide. At present, a large number of researches have already reported that certain plant-derived allelochemicals can inhibit weed growth. In this study, the allelopathic effect of Artemisia argyi was investigated via a series of laboratory experiments and field trial. Firstly, water-soluble extracts exhibited the strongest allelopathic inhibitory effects on various plants under incubator conditions, after the different extracts authenticated by UPLC-Q-TOF-MS. Then, the allelopathic effect of the A. argyi was systematacially evaluated on the seed germination and growth of Brassica pekinensis, Lactuca sativa, Oryza sativa, Portulaca oleracea, Oxalis corniculata and Setaria viridis in pot experiments, it suggested that the A. argyi could inhibit both dicotyledons and monocotyledons not only by seed germination but also by seedling growth. Furthermore, field trial showed that the A. argyi significantly inhibited the growth of weeds in Chrysanthemum morifolium field with no adverse effect on the growth of C. morifolium. At last, RNA-Seq analysis and key gene detection analysis indicated that A.argyi inhibited the germination and growth of weed via multi-targets and multi-paths while the inhibiting of chlorophyll synthesis of target plants was one of the key mechanisms. In summary, the A. argyi was confirmed as a potential raw material for the development of preventive herbicides against various weeds in this research. Importantly, this discovery maybe provide scientific evidence for the research and development of environmentally friendly herbicides in the future.

Re-Analysis of Abdominal Gland Volatilome Secretions of the African Weaver Ant, Oecophylla longinoda (Hymenoptera: Formicidae).

The African weaver ant, Oecophylla longinoda, is used as a biological control agent for the management of pests. The ant has several exocrine glands in the abdomen, including Dufour's, poison, rectal, and sternal glands, which are associated with pheromone secretions for intra-specific communication. Previous studies have analyzed the gland secretions of Dufour's and poison glands. The chemistry of the rectal and sternal glands is unknown. We re-analyzed the secretions from Dufour's and poison glands plus the rectal and sternal glands to compare their chemistries and identify additional components. We used the solid-phase microextraction (SPME) technique to collect gland headspace volatiles and solvent extraction for the secretions. Coupled gas chromatography-mass spectrometry (GC-MS) analysis detected a total of 78 components, of which 62 were being reported for the first time. These additional components included 32 hydrocarbons, 12 carboxylic acids, 5 aldehydes, 3 alcohols, 2 ketones, 4 terpenes, 3 sterols, and 1 benzenoid. The chemistry of Dufour's and poison glands showed a strong overlap and was distinct from that of the rectal and sternal glands. The different gland mixtures may contribute to the different physiological and behavioral functions in this ant species.

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production.

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Clock genes and environmental cues coordinate Anopheles pheromone synthesis, swarming, and mating.

Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period (per) and timeless (tim) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity.

Biosynthesis of aphid alarm pheromone is modulated in response to starvation stress under regulation by the insulin, glycolysis and isoprenoid pathways.

The mechanism for biosynthesis and molecular regulation of the aphid alarm pheromone (AAP) is still a mystery. Previous studies indicated that the biosynthesis of AAP was directly affected by the terpenoid backbone biosynthesis pathway, and several pathways involved in nutritional metabolism providing the bricks for AAP biosynthesis were up-regulated in response to simulated stimulation. This suggests that AAP biosynthesis might be regulated by complex metabolic pathways. Here the molecular responses of the bird cherry-oat aphid Rhopalosiphum padi to starvation stress were investigated, and the molecular pathways were further analyzed by using RNA interference (RNAi) and protein inhibitor, combined with gas chromatography-mass spectrometry analysis of (E)-ß-farnesene (EßF), the major component of the alarm pheromone in R. padi. The results showed that the nutritional stress significantly reduced the weight of aphid and the quantity of EßF, and meanwhile dramatically up-regulated the insulin receptor genes (InsR1/2) and down-regulated the downstream genes encoding the kinases PI3K and Akt, key enzymes in the glycolysis pathway (HK, A6PFK, PK) and the isoprenoid pathway (ACSS, HMGR, FPPS1, FPPS2, GGPPS, DPPS). PI3K inhibitor LY294002 treatment and RNAi-mediated knockdown of InsR1/2 significantly reduced the expression level of downstream genes and the quantity of EßF. Furthermore, knockdown of PK, the rate-limiting enzyme in the glycolysis pathway, down-regulated the genes in the isoprenoid pathway and the production of EßF; knockdown of the genes encoding isoprenyl diphosphate enzymes revealed that FPPS1 and FPPS2 were both required for EßF biosynthesis. Our data suggested that AAP is synthesized via glycolysis and isoprenoid pathways under regulation by the insulin signaling pathway.

Sex pheromone biosynthesis, storage and release in a female moth: making a little go a long way.

Moth pheromone research has pioneered much of our understanding of long-distance chemical communication. Two important characteristics of this communication have, however, remained largely unaddressed: the release of small quantities of pheromone by most moth species, despite potential advantages of releasing greater amounts, and the intermittency of release in some species, limiting the time of mate attraction. We addressed the proximate mechanisms underlying these characteristics by manipulating biosynthesis, storage and release of pheromone in females of the noctuid moth Chloridea virescens. We found that (i) mass release is determined by pheromone mass on the gland surface; (ii) amounts synthesized are limited by pheromone biosynthesis activating neuropeptide concentration, not precursor availability; (iii) some gland structural feature limits mass release rate; (iv) intermittent calling enables release at a mass rate greater than biosynthetic rate; and (v) at typical mass release rates, the periodicity of pheromone availability on the gland surface roughly matches the periodicity (intermittency) of calling. We conclude that mass release in C. virescens and possibly many other species is low because of constraints on biosynthesis, storage and gland structure. Further, it appears the behaviour of intermittent calling in C. virescens may have evolved as a co-adaptation with pheromone availability, allowing females to release pheromone intermittently at higher mass rates than the biosynthesis rate.

Ratiometric quorum sensing governs the trade-off between bacterial vertical and horizontal antibiotic resistance propagation.

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies.

Pheromonal activities of the bombykol isomer, (10E,12E)-10,12-hexadecadien-1-ol, in the pheromone gland of the silkmoth Bombyx mori.

Bombykol (EZ) is the single component of the female sex pheromone in the silkmoth Bombyx mori. EZ alone evokes full courtship behaviors from conspecific males; however, its geometric isomer (EE) was consistently detected in the pheromone glands (PG) of 16 B. mori strains and a field population of the wild silkmoth Bombyx mandarina, which also uses EZ as the single pheromone component. We investigated the pheromonal activities of EE using a commercial hybrid strain of B. mori, Kinshu × Showa. The behavioral assay demonstrated that a 104-105-fold higher dose of EE than EZ was able to elicit behavioral responses from males. To elucidate whether the trace contaminant of EZ in the EE standard is responsible for these responses, we examined the responses of male antennae to EE using a gas chromatograph-electroantennographic detector system (GC-EAD). The EE, at high doses elicited marginal responses from the male antennae. We next examined antennal and behavioral responses of B. mori whose BmOR1 gene, which is responsible for the reception of bombykol, was knocked out. The knockout of BmOR1 resulted in the complete loss of antennal and behavioral responses to EE and EZ, demonstrating that if EE itself is active, it induces these responses via the incidental stimulation of BmOR1, not via the stimulation of EE-specific receptors. The existence of EE in the PG of B. mori and B. mandarina is discussed from the viewpoints of pheromone biosynthesis and the evolution of pheromone communication systems.

Do Bumble Bees Produce Brood Pheromones?

Reproductive division of labor, a defining feature of social insects, is often regulated by a combination of behavioral and chemical means. It is hypothesized that behavioral interactions play a more important role in regulating reproduction of primitive eusocial species, while pheromones are typically used by large sized, advanced eusocial species. Here we examined if worker reproduction in the primitively eusocial species Bombus impatiens is regulated by brood pheromones. We recently demonstrated that worker egg laying in this species is inhibited by young larvae and triggered by pupae. However, the mechanism by which the brood communicates its presence and whether brood or hunger pheromones are involved remain unknown. We found that workers were behaviorally attracted to pupae over larvae or control in a choice experiment, in line with their reproductive interests. However, odors from larvae or pupae were insufficient to inhibit worker reproduction. We further show that the youngest larvae are particularly vulnerable to starvation, however, despite a slight attraction and fewer eggs laid by workers in the presence of starved compared with fed larvae, these effects were insignificant. Our study demonstrates that workers can differentiate between larvae and pupae, but not between starved and fed larvae based on olfactory information. However, these signals alone do not explain the reduction in worker egg laying previously found. Bumble bee workers may use information from multiple sources or rely solely on behavioral interactions with brood and other females to make decisions about reproduction, in line with their small colony size and simple social organization.

Hydroxylation patterns associated with pheromone synthesis and composition in two honey bee subspecies Apis mellifera scutellata and A. m. capensis laying workers.

Colony losses due to social parasitism in the form of reproductive workers of the Apis mellifera capensis clones results from the production of queen-like pheromonal signals coupled with ovarian activation in these socially parasitic honey bees. While the behavioral attributes of these social parasites have been described, their genetic attributes require more detailed exploration. Here, we investigate the production of mandibular gland pheromones in queenless workers of two sub-species of African honey bees; A. m. scutellata (low reproductive potential) and A. m. capensis clones (high reproductive potential). We used standard techniques in gas chromatography to assess the amounts of various pheromone components present, and qPCR to assess the expression of cytochrome P450 genes cyp6bd1 and cyp6as8, thought to be involved in the caste-dependent hydroxylation of acylated stearic acid in queens and workers, respectively. We found that, for both subspecies, the quality and quantity of the individual pheromone components vary with age, and that from the onset, A. m. capensis parasites make use of gene pathways typically upregulated in queens in achieving reproductive dominance. Due to the high production of 9-hydroxy-decenoic acid (9-HDA) the precursor to the queen substance 9-oxo-decenoic acid (9-ODA) in newly emerged capensis clones, we argue that clones are primed for parasitism upon emergence and develop into fully fledged parasites depending on the colony's social environment.

The Differential Expression of Mevalonate Pathway Genes in the Gut of the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Is Unrelated to the de Novo Synthesis of Terpenoid Pheromones.

Bark beetles commonly produce de novo terpenoid pheromones using precursors synthesized through the mevalonate pathway. This process is regulated by Juvenile Hormone III (JH III). In this work, the expression levels of mevalonate pathway genes were quantified after phloem feeding-to induce the endogenous synthesis of JH III-and after the topical application of a JH III solution. The mevalonate pathway genes from D. rhizophagus were cloned, molecularly characterized, and their expression levels were quantified. Also, the terpenoid compounds produced in the gut were identified and quantified by Gas Chromatography Mass Spectrometry (GC-MS). The feeding treatment produced an evident upregulation, mainly in acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IPPI) genes, and males reached higher expression levels compared to females. In contrast, the JH III treatment did not present a clear pattern of upregulation in any sex or time. Notably, the genes responsible for the synthesis of frontalin and ipsdienol precursors (geranyl diphosphate synthase/farnesyl diphosphate synthase (GPPS/FPPS) and geranylgeranyl diphosphate synthase (GGPPS)) were not clearly upregulated, nor were these compounds further identified. Furthermore, trans-verbenol and myrtenol were the most abundant compounds in the gut, which are derived from an α-pinene transformation rather than de novo synthesis. Hence, the expression of mevalonate pathway genes in D. rhizophagus gut is not directed to the production of terpenoid pheromones, regardless of their frequent occurrence in the genus Dendroctonus.

Gene Modules Co-regulated with Biosynthetic Gene Clusters for Allelopathy between Rice and Barnyardgrass.

Allelopathy is a central process in crop-weed interactions and is mediated by the release of allelochemicals that result in adverse growth effects on one or the other plant in the interaction. The genomic mechanism for the biosynthesis of many critical allelochemicals is unknown but may involve the clustering of non-homologous biosynthetic genes involved in their formation and regulatory gene modules involved in controlling the coordinated expression within these gene clusters. In this study, we used the transcriptomes from mono- or co-cultured rice and barnyardgrass to investigate the nature of the gene clusters and their regulatory gene modules involved in the allelopathic interactions of these two plants. In addition to the already known biosynthetic gene clusters in barnyardgrass we identified three potential new clusters including one for quercetin biosynthesis and potentially involved in allelopathic interaction with rice. Based on the construction of gene networks, we identified one gene regulatory module containing hub transcription factors, significantly positively co-regulated with both the momilactone A and phytocassane clusters in rice. In barnyardgrass, gene modules and hub genes co-expressed with the gene clusters responsible for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) biosynthesis were also identified. In addition, we found three genes in barnyardgrass encoding indole-3-glycerolphosphate synthase that regulate the expression of the DIMBOA cluster. Our findings offer new insights into the regulatory mechanisms of biosynthetic gene clusters involved in allelopathic interactions between rice and barnyardgrass, and have potential implications in controlling weeds for crop protection.

Identification and Gene Expression Analysis of the Pheromone Biosynthesis Activating Neuropeptide Receptor (PBANR) From the Ostrinia furnacalis (Lepidoptera: Pyralidae).

Pheromonal communication is important in insect mate finding and reproduction. Identifying components of pest insect pheromone system is a first step to disrupt pest insect reproduction. In this study, we identified and cloned the pheromone biosynthesis activating neuropeptide receptor (PBANR) from the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), which is one of the most damaging pests of corn and other crops in parts of Asia and Australia. The O. furnacalis PBANR (OstfuPBANR) gene has an ORF of 1,086 bp and encoded 362 amino acids with seven transmembrane domains and had a high sequence identity to known lepidopteran PBANRs. Expression analysis showed that OstfuPBANR was highly expressed in the pheromone glands compared with other tissues, consistent with other studies. Interestingly, OstfuPBANR was expressed higher in the larval stages compared to the pupal or adult stages, suggesting that OstfuPBANR may have broad functions in larva beyond adult pheromone synthesis.

Production of alarm pheromone starts at embryo stage and is modulated by rearing conditions and farnesyl diphosphate synthase genes in the bird cherry-oat aphid Rhopalosiphum padi.

The major component of aphid alarm pheromone is (E)-ß-farnesene (EßF), but the molecular mechanisms of EßF synthesis are poorly understood. Here we established a biological model to study the modulation of EßF synthesis in the bird cherry-oat aphid Rhopalosiphum padi by using quantitative polymerase chain reaction, gas chromatography/mass spectrometry and RNA interference. Our results showed that the rearing conditions significantly affected the weight of adult and modulated EßF synthesis in a transgenerational manner. Specifically, the quantity of EßF per milligram of aphid was significantly reduced in the individually reared adult or 1st-instar nymphs derived from 1-day-old adult reared individually, but EßF in the nymph derived from 2-day-old adult that experienced collective conditions returned to normal. Further study revealed that the production of EßF started in embryo and was extended to early nymphal stage, which was modulated by farnesyl diphosphate synthase genes (RpFPPS1 and RpFPPS2) and rearing conditions. Knockdown of RpFPPS1 and RpFPPS2 confirmed the role played by FPPS in the biosynthesis of aphid alarm pheromone. Our results suggested that the production of EßF starts at the embryo stage and is modulated by FPPS and rearing conditions in R. padi, which sheds lights on the modulatory mechanisms of EßF in the aphid.