Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Natural variation in maize aphid resistance is associated with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity.

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Stable isotope studies reveal pathways for the incorporation of non-essential amino acids in Acyrthosiphon pisum (pea aphids).

Plant roots incorporate inorganic nitrogen into the amino acids glutamine, glutamic acid, asparagine and aspartic acid, which together serve as the primary metabolites of nitrogen transport to other tissues. Given the preponderance of these four amino acids, phloem sap is a nutritionally unbalanced diet for phloem-feeding insects. Therefore, aphids and other phloem feeders typically rely on microbial symbionts for the synthesis of essential amino acids. To investigate the metabolism of the four main transport amino acids by the pea aphid (Acyrthosiphon pisum), and its Buchnera aphidicola endosymbionts, aphids were fed defined diets with stable isotope-labeled glutamine, glutamic acid, asparagine or aspartic acid (U-(13)C, U-(15)N; U-(15)N; α-(15)N; or γ-(15)N). The metabolic fate of the dietary (15)N and (13)C was traced using gas chromatography-mass spectrometry (GC-MS). Nitrogen was the major contributor to the observed amino acid isotopomers with one additional unit mass (M+1). However, there was differential incorporation, with the amine nitrogen of asparagine being incorporated into other amino acids more efficiently than the amide nitrogen. Higher isotopomers (M+2, M+3 and M+4) indicated the incorporation of varying numbers of (13)C atoms into essential amino acids. GC-MS assays also showed that, even with an excess of dietary labeled glutamine, glutamic acid, asparagine or aspartic acid, the overall content of these amino acids in aphid bodies was mostly the product of catabolism of dietary amino acids and subsequent re-synthesis within the aphids. Thus, these predominant dietary amino acids are not passed directly to Buchnera endosymbionts for synthesis of essential amino acids, but are rather are produced de novo, most likely by endogenous aphid enzymes.

Matching the supply of bacterial nutrients to the nutritional demand of the animal host.

Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.

Collision-induced dissociation mass spectra of glucosinolate anions.

Collision-induced dissociation (CID) mass spectra of differently substituted glucosinolates were investigated under negative-ion mode. Data obtained from several glucosinolates and their isotopologues ((34)S and (2)H) revealed that many peaks observed are independent of the nature of the substituent group. For example, all investigated glucosinolate anions fragment to produce a product ion observed at m/z 195 for the thioglucose anion, which further dissociates via an ion/neutral complex to give two peaks at m/z 75 and 119. The other product ions observed at m/z 80, 96 and 97 are characteristic for the sulfate moiety. The peaks at m/z 259 and 275 have been attributed previously to glucose 1-sulfate anion and 1-thioglucose 2-sulfate anion, respectively. However, based on our tandem mass spectrometric experiments, we propose that the peak at m/z 275 represents the glucose 1-thiosulfate anion. In addition to the common peaks, the spectrum of phenyl glucosinolate (beta-D-Glucopyranose, 1-thio-, 1-[N-(sulfooxy)benzenecarboximidate] shows a substituent-group-specific peak at m/z 152 for C(6)H(5)-C(=NOH)S(-), the CID spectrum of which was indistinguishable from that of the anion of synthetic benzothiohydroxamic acid. Similarly, the m/z 201 peak in the spectrum of phenyl glucosinolate was attributed to C(6)H(5)-C(=S)OSO(2)(-).

Isothiocyanates stimulating oviposition by the diamondback moth, Plutella xylostella.

Recognition of cabbage as a host plant for the diamondback moth (DBM) has previously been shown to depend on compounds that are extracted by soaking intact foliage in chloroform. Analysis of such chloroform extracts by open column chromatography has now resulted in the isolation of highly active fractions that elicit oviposition on treated filter papers. Further separation of these fractions by high-performance liquid chromatography revealed the presence of two distinct groups of active compounds that may be classified as volatile and non-volatile. The two prominent volatile components were separated and identified by mass spectrometry as the isothiocyanates, iberin (3-methylsulfinylpropyl isothiocyanate) and sulforaphane (4-methylsulfinyl-3-butenyl isothiocyanate). Subsequent bioassays of a range of isothiocyanates showed that iberin and sulforaphane were the most active of those tested. Other isothiocyanates with sulfur in the side chain were also active, whereas alkyl and phenyl isothiocyanates had only limited activity. In electrophysiological experiments, electroantennograms (EAGs) indicated positive responses of moth antennae to the isothiocyanates that were most active in behavioral assays. Since sulforaphane has been identified as a major inducer of anticarcinogenic activity in mouse tissue, a synthetic analog (exo-2-acetyl-5-isothiocyanatonorbornane) that shows similar inducer activity was tested on DBM. This bicyclic analog was highly active in both behavioral and EAG assays, suggesting similarity in receptor sites for the two types of biological activity.

Combined roles of contact stimulant and deterrents in assessment of host-plant quality by ovipositing zebra swallowtail butterflies.

Zebra swallowtail (Eurytides marcellus) butterflies are stimulated to oviposit by a single compound, 3-caffeoyl-muco-quinic acid (1). Analysis of the aqueous extracts of the leaves of the host. Asimina triloba, showed that they contained stimulant 1, its isomer (2), and the flavonoids rutin (3) and nicotiflorine (4) as major components. We compared the concentrations of compounds 1-4 in terminal leaves (TL) and expanded leaves (EL) of the host plants at four different times throughout the growing season. In spring, the concentration of 1 was highest in TLs, and flavonoids were not detectable or present at low levels. As the season progressed, however, the concentrations of flavonoids increased, reached maxima by late summer, and then decreased as the plants started senescing. There were also significant differences in the concentrations of these compounds between TLs and ELs. In a choice assay with model leaves, we tested equivalent amounts of post-dichloromethane aqueous extracts made in spring (May) and in fall (September). September extracts received significantly fewer approaches and eggs. In greenhouse experiments with potted A. triloba plants, the butterflies chose some leaves to lay eggs, while others were rejected or ignored. Analyses showed that the concentrations of compound 1 were not significantly different in the three kinds of leaves. The flavonoids (3 and 4), however, were significantly higher in the leaves that were ignored. Multiple-choice tests using model plants suggested that concentrations of both flavonoids and stimulant were important in assessing host suitability. There was a gradual decrease in approaches as the concentration of 1 decreased. Higher amounts of flavonoids deterred egg laying even in the presence of high concentrations of stimulant 1. At lower concentrations of 1, the addition of low doses of flavonoids deterred egg laying. Thus, the results suggest that the butterflies use both qualitative and quantitative information about these compounds to assess host quality. This behavior may have evolved to take advantage of seasonal variation in the chemistry of their host, A. triloba.