Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Plant growth-promoting rhizobacteria modulate induced corn defense against Spodoptera litura (Lepidoptera: Noctuidae).

Common cutworm, Spodoptera litura is an important pest of corn causing significant crop yield loss. Synthetic insecticides have mostly been used to combat this pest, raising human and environmental health concerns. Plant growth-promoting rhizobacteria (PGPR) could compensate for or augment the harmful effects of agrochemicals. Herein, we aimed to assess whether PGPR-induced defenses in corn plants impact the host-plant selection behavior of S. litura. Headspace volatile organic compounds were analyzed using gas chromatography-mass spectrometry. Larvae fed inoculated corn exhibited lower weights and relative growth rate than noninoculated plants. Under choice experiments, PGPR-treated plants significantly reduced percentage leaf damage area and oviposition rate compared to untreated plants. Volatile organic compound ratio emission varied significantly between control and PGPR treatments, which, in part, explains feeding and oviposition deterrence in PGPR-treated plants. The results demonstrate that PGPR inoculation can enhance corn resistance to S. litura, making it a promising candidate for crop protection strategies.

Intrinsic Factors Associated with Dietary Toxin Quantity and Concentration in the Nuchal Glands of a Natricine Snake Rhabdophis Tigrinus.

The snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads and stores them in the nuchal glands as defensive toxins. It has previously been shown that there are individual differences in the total quantity of BDs stored in the nuchal glands of adult R. tigrinus and that BD quantities and profiles of R. tigrinus exhibit geographic variation. However, no previous study has examined the total quantity of BDs as a percentage of body mass (relative BD quantity) and the concentration of BDs in the nuchal gland fluid (BD gland concentration). In addition, intrinsic factors that are associated with relative BD quantity and BD concentration have not been examined within a single population. We collected 158 adult snakes from an area of central Japan from May to October and analyzed their BD quantities by UV analysis. We assessed individual differences in BD quantity, relative BD quantity and BD gland concentration. We found that 1) in approximately 60% of the 158 individuals, the BD gland concentration was greater than 50%; 2) body length and body condition are positively correlated with relative BD quantity and BD gland concentration; 3) even in a single population, individual differences of BD quantity are large, and are greater in females than in males; and 4) relative BD quantity and BD gland concentration of females during the gestation season are lower than those during the non-gestation season.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Absolute Stereochemistry of TXIB, a Bioactive Plasticizer That Inhibits Oviposition of the Peach Fruit Moth, Carposina sasakii (Lepidoptera: Carposinidae).

TXIB (2,2,4-trimethyl-1,3-pentanediol diisobutyrate), a common plasticizer, inhibits the peach fruit moth, Carposina sasakii from ovipositing on young apple fruits. Using an insect assay and gas chromatography with an electroantennographic detector (GC-EAD) analysis, the stereochemistry of the bioactive compound was identified to be 3R, whereas (3S)-TXIB showed no activity. The inactive enantiomer did not inhibit the activity of commercially available TXIB, which was a racemic mixture. When the female moth was placed on the TXIB-treated fruits in the oviposition assay, she repeated grooming antennae. The reason why the moth detects this artificial compound is unknown, although the stereochemical structure of TXIB could have given an abnormality to the moth's antennae.

A nonproteinogenic amino acid, ß-tyrosine, accumulates in young rice leaves via long-distance phloem transport from mature leaves.

Oryza sativa L. ssp. japonica cv. Nipponbare produces a nonproteinogenic amino acid (3R)-ß-tyrosine from l-tyrosine by tyrosine aminomutase (OsTAM1). However, physiological and ecological function(s) of ß-tyrosine have remained obscure. Often an improved understanding of metabolite localization and transport can aid in design of experiments to test physiological functions. In the current study, we investigated the distribution pattern of ß-tyrosine in rice seedlings and found that ß-tyrosine is most abundant in the youngest leaves. Based upon observations of high TAM1 activity in mature leaves, we hypothesized that ß-tyrosine is transported from mature leaves to young leaves. Patterns of predominant mature synthesis and young leaf accumulation were supported by stable isotope studies using labeled ß-tyrosine and the removal of mature leaves. Stem exudate analyses was also consistent with ß-tyrosine transport through phloem. Thus, we identify young leaves as a key target in efforts to understand the biological function(s) of ß-tyrosine in rice.

New Insights Into Dietary Toxin Metabolism: Diversity in the Ability of the Natricine Snake Rhabdophis tigrinus to Convert Toad-Derived Bufadienolides.

The Japanese natricine snake Rhabdophis tigrinus sequesters cardiotonic steroids, bufadienolides (BDs), from ingested toads in the nuchal glands as defensive toxins. A previous study showed that R. tigrinus in captivity converts dietary BDs when it sequesters them. However, it is unknown whether the dietary BDs are actually converted and the modified products accumulated under natural conditions. It is also unknown to what extent the BD profile of ingested toads is reflected in that of the snake. We collected 123 snakes from throughout Japan, analyzed their BD profiles by liquid chromatography/mass spectrometry, and identified 15 BDs from R. tigrinus by nuclear magnetic resonance analyses. We also compared their BD profiles using hierarchical cluster analysis (HCA). HCA exhibited two main clusters associated with their collection locations: eastern and western regions of the Japanese main islands. These results, coupled with previous findings on the BDs of Japanese toads, suggest that 1) R. tigrinus converts toad-derived BDs into other compounds under natural conditions; 2) there are both universal and regionally-specific conversions of dietary BDs by R. tigrinus; and 3) geographic variation in toad BD profiles is partially reflected in the variation of snake BD profiles.

Triterpenes induced by young apple fruits in response to herbivore attack.

Apples Malus domestica, known as a rich source of triterpene acids, induced more variety and quantity of triterpene acids in response to herbivory or mechanical damage. There were 3 major induced compounds: pomaceic acid and euscaphic acid, both of which are known apple triterpene acids, and 2α,19α-dihydroxy-3-oxours-12-en-28-oic acid (named eriobotoric acid), which was first identified in apples. In this study, the 3 compounds' induction curves after damage, varietal differences in induction amounts, and physiological roles against pest insects were further investigated. Eriobotoric acid showed clear antifeedant activity against lepidopteran insect Spodoptera litura but not against apple pests.

Variation in Bufadienolide Composition of Parotoid Gland Secretion From Three Taxa of Japanese Toads.

Toads of the genus Bufo synthesize and accumulate bufadienolides (BDs) in their parotoid glands. BDs are cardiotonic steroids that play an important role in defense against the toads' predators. Three bufonid taxa occur in mainland Japan, Bufo japonicus formosus, B. j. japonicus, and B. torrenticola. The chemical structures of BDs isolated from B. j. formosus were studied several decades ago, but there is no further information on the toxic components of Japanese toads and their metabolism. In this study, we analyzed BDs of toads from throughout Japan and compared the BD profiles by liquid chromatography/mass spectrometry (LC/MS) and hierarchical cluster analysis (HCA). We observed BDs in three taxa of Japanese toads, and identified five of the most common BDs by nuclear magnetic resonance (NMR) analyses. Of the five BDs, only bufalin was detected in all individuals. HCA of individual BD profiles divided the three taxa into five primary clusters and several subclusters. This result indicates that BD profiles differ both among and within the taxa. The clustering pattern of BDs is generally concordant with a phylogenetic tree reconstructed from the mitochondrial cytochrome b gene of Japanese toads. Our results suggest that the BDs of Japanese toads have diversified not in response to specific selective pressures, but simply due to population structuring over evolutionary time.

An easy, inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing.

Chitin, poly (ß-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan-I3- complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05-0.5 µg/µL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers. Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes.

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H-2CO]+ ions derived from isoflavones and [M+H-B-ring-CO]+ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1',2',3',4',5',6',2,3,4-13C9] and [2',3',5',6',2-D5] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied 13C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H-2CO]+ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H-B-ring-CO]+ ion is demonstrated to contain a C2H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSn.

Inducible De Novo Biosynthesis of Isoflavonoids in Soybean Leaves by Spodoptera litura Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS.

Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[13C9, 15N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in 13C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), 13C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and 13C-labeled isoflavone 7-O-(6″-O-malonyl-ß-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of 13C-labeled flavones and flavonol (4',7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-ß-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.

Physiological function and ecological aspects of fatty acid-amino acid conjugates in insects.

In tritrophic interactions, plants recognize herbivore-produced elicitors and release a blend of volatile compounds (VOCs), which work as chemical cues for parasitoids or predators to locate their hosts. From detection of elicitors to VOC emissions, plants utilize sophisticated systems that resemble the plant-microbe interaction system. Fatty acid-amino acid conjugates (FACs), a class of insect elicitors, resemble compounds synthesized by microbes in nature. Recent evidence suggests that the recognition of insect elicitors by an ancestral microbe-associated defense system may be the origin of tritrophic interactions mediated by FACs. Here we discuss our findings in light of how plants have customized this defense to be effective against insect herbivores, and how some insects have successfully adapted to these defenses.

N-(18-hydroxylinolenoyl)-L-glutamine: a newly discovered analog of volicitin in Manduca sexta and its elicitor activity in plants.

Plants attacked by insect herbivores release a blend of volatile organic compounds (VOCs) that serve as chemical cues for host location by parasitic wasps, natural enemies of the herbivores. Volicitin, N-(17-hydroxylinolenoyl)-L-glutamine, is one of the most active VOC elicitors found in herbivore regurgitants. Our previous study revealed that hydroxylation on the 17th position of the linolenic acid moiety of N-linolenoyl-L-glutamine increases by more than three times the elicitor activity in corn plants. Here, we identified N-(18-hydroxylinolenoyl)-L-glutamine (18OH-volicitin) from larval gut contents of tobacco hornworm (THW), Manduca sexta. Eggplant and tobacco, two solanaceous host plants of THW larvae, and corn, a non-host plant, responded differently to this new elicitor. Eggplant and tobacco seedlings emitted twice the amount of VOCs when 18OH-volicitin was applied to damaged leaf surfaces compared to N-linolenoyl-L-glutamine, while both these fatty acid amino acid conjugates (FACs) elicited a similar response in corn seedlings. In both solanaceous plants, there was no significant difference in the elicitor activity of 17OH- and 18OH-volicitin. Interestingly, other lepidopteran species that have 17OH-type volicitin also attack solanaceous plants. These data suggest that plants have developed herbivory-detection systems customized to their herbivorous enemies.

Children's knowledge of hierarchical phrase structure: quantifier floating in Japanese.

The interpretation of floating quantifiers in Japanese requires knowledge of hierarchical phrase structure. However, the input to children is insufficient or even misleading, as our analysis indicates. This presents an intriguing question on learnability: do children interpret floating quantifiers based on a structure-dependent rule which is not obvious in the input or do they employ a sentence comprehension strategy based on the available input? Two experiments examined four- to six-year-old Japanese-speaking children for their interpretations of floating quantifiers in SOV and OSV sentences. The results revealed that no child employed a comprehension strategy in terms of the linear ordering of constituents, and most five- and six-year-olds correctly interpreted floating quantifiers when word-order difficulty was reduced. These facts indicate that children's interpretation of floating quantifiers is structurally dependent on hierarchical phrase structure, suggesting that this knowledge is a part of children's grammar despite the insufficient input available to them.

Active role of fatty acid amino acid conjugates in nitrogen metabolism in Spodoptera litura larvae.

Since the first fatty acid amino acid conjugate (FAC) was isolated from regurgitant of Spodoptera exigua larvae in 1997 [volicitin: N-(17-hydroxylinolenoyl)-L-glutamine], their role as elicitors of induced responses in plants has been well documented. However, studies of the biosyntheses and the physiological role of FACs in the insect have been minimal. By using (14)C-labeled glutamine, glutamic acid, and linolenic acid in feeding studies of Spodoptera litura larvae, combined with tissue analyses, we found glutamine in the midgut cells to be a major source for biosynthesis of FACs. Furthermore, 20% of the glutamine moiety of FACs was derived from glutamic acid and ammonia through enzymatic reaction of glutamine synthetase (GS). To determine whether FACs improve GS productivity, we studied nitrogen assimilation efficiency of S. litura larvae fed on artificial diets containing (15)NH(4)Cl and glutamic acid. When the diet was enriched with linolenic acid, the nitrogen assimilation efficiency improved from 40% to >60%. In the lumen, the biosynthesized FACs are hydrolyzed to fatty acids and glutamine, which are reabsorbed into tissues and hemolymph. These results strongly suggested that FACs play an active role in nitrogen assimilation in Lepidoptera larva and that glutamine containing FACs in the gut lumen may function as a form of storage of glutamine, a key compound of nitrogen metabolism.

Evaluation of antixenosis in soybean against Spodoptera litura by dual-choice assay aided by a statistical analysis model: Discovery of a novel antixenosis in Peking.

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd-5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd-5th instar larvae than for 2nd instar.

Fatty acid-amino acid conjugates diversification in lepidopteran caterpillars.

Fatty acid amino acid conjugates (FACs) have been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and its biochemical precursor, N-linolenoyl-L-glutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-L-glutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species.

Inhibitory substances contained in calcium carbonate wettable powder on the oviposition of the peach fruit moth, Carposina sasakii.

Spraying a calcium carbonate suspension "White Coat" on the fruit of apples significantly suppresses the oviposition of the peach fruit moth, Carposina sasakii. In gas chromatography (GC) with an electroantennographic detector analysis, adult female antennae showed responses to three compounds that were identified as 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (TXIB) and its two mono-hydrolyzed analogs, texanols (1- and 3-isobutyrates), all added as a plasticizer to the agents. An oviposition-choice test using adult moths revealed that TXIB has clear deterrent properties when applied to young apple fruits. Video recording analysis showed that female moths spent longer on self-grooming and searching around TXIB-treated fruits. In the same assay, pure calcium carbonate treatment prevented the moths from climbing up or landing on the fruits, while such was not the case with White Coat-treated fruits. TXIB, an adjuvant aimed to provide rain/wind resistance, weakened the slipperiness of the calcium carbonate coating but, coincidentally, maintained the oviposition inhibitory activity of the White Coat by its deterrent odorant.

Efficient incorporation of free oxygen into volicitin in Spodoptera litura common cutworm larvae.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] has previously been identified from the lepidopteran larval regurgitant as an elicitor of plant volatile emission. The efficient incorporation of free oxygen into volicitin by Spodoptera litura larvae is demonstrated here by rearing them under (18)O(2) for three days. (18)O-labeling of the hydroxyl group of volicitin was confirmed by liquid chromatography/mass spectrometry-ion trap-time-of-flight (LC/MS-IT-TOF) and suggests the activity of a monooxygenase in volicitin biosynthesis.