Factors Influencing Responses to Alarm Pheromone by Larvae of Invasive Cane Toads, <Emphasis Type="Italic">Bufo marinus</Emphasis>

If pheromonal communication systems of invasive species differ from those of native biota, it may be possible to control the invader by exploiting that difference. When injured, the larvae of cane toads, Bufo marinus, an invasive species of major concern in tropical Australia, produce species-specific chemical cues that alert conspecific tadpoles to danger. Repeated exposure to the alarm chemical reduces tadpole survival rates and body sizes at metamorphosis and, thus, could help control toad populations. To evaluate the feasibility of this approach, we need to know how the intensity of toad tadpole response to the alarm chemical is affected by factors such as water temperature, time of day, larval stage and feeding history, geographic origin of the tadpoles, and habituation. Information on these topics may enable us to optimize deployment, so that tadpoles encounter pheromone at the times and places that confer maximum effect. In our studies, tadpole density, nutritional state, larval stage, and geographic origin had little effect on the intensity of the alarm response, but tadpoles reacted most strongly in higher water temperatures and during daylight hours. Repeated, once-daily exposure to pheromone did not induce habituation, but repeated exposure at 15-min intervals did not elicit further responses after 2 h total exposure. The insensitivity of response to most factors tested means that the effectiveness of the pheromone as a control agent should be relatively robust.     

Variation in Chemical Defense Among Natural Populations of Common Toad, <Emphasis Type="Italic">Bufo bufo</Emphasis>, Tadpoles: the Role of Environmental Factors

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles’ toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.     

Shifts in Life History as a Response to Predation in Western Toads (Bufo boreas)

Larval western toads (Bufo boreas) are known to exhibit antipredator behavior in response to both chemical alarm cues released from injured conspecifics and chemical cues of predatory invertebrates. In this study, we tested whether long-term exposure to predator and alarm cues resulted in an adaptive shift in life history characteristics of the toads. We raised groups of tadpoles in the presence of: (1) predatory backswimmers (Notonecta spp.) that were fed toad tadpoles, (2) nonpredatory water boatman (Corixidae), and (3) chemical alarm cues of injured conspecifics. Tadpoles raised in the presence of both chemical alarm cues and cues of predators fed tadpoles metamorphosed in significantly shorter time than those raised in the presence of the nonpredator control. Reducing time taken to reach metamorphosis would reduce exposure to aquatic predators. There was no difference among treatments in the size at metamorphosis. Our results suggest that this shift in metamorphic characteristics may represent a facultative alteration in life history.     

Sex Pheromone Components of the Pear Fruit Moth, <Emphasis Type="Italic">Acrobasis pyrivorella</Emphasis> (Matsumura)

We analyzed the sex pheromone of the pear fruit moth, Acrobasis pyrivorella, by means of gas chromatography–electroantennographic detection (GC-EAD) and GC–mass spectrometry. Two EAD-active compounds were detected in the pheromone gland extract of females. They were identified as (Z)-9-pentadecenyl acetate (Z9-15:OAc) and pentadecyl acetate (15:OAc). The amounts per female gland (mean ± standard error) of these compounds were 12.9 ± 2.8 and 0.8 ± 0.1 ng, respectively. Synthetic Z9-15:OAc (300 μg) attracted conspecific males in field trapping experiments. When 15:OAc (21 μg; 7% of Z9-15:OAc quantity) was added, the number of males trapped increased significantly. Catch in traps baited with the mixture of these compounds was greater than that in traps baited with 1–3-day-old virgin females. We, therefore, conclude that Z9-15:OAc and 15:OAc are sex pheromone components of this species.     

Fumigant and Contact Toxicities of Monoterpenes to <Emphasis Type="Italic">Sitophilus oryzae</Emphasis> (L.) and <Emphasis Type="Italic">Tribolium castaneum</Emphasis> (Herbst) and their Inhibitory Effects on Acetylcholinesterase Activity

A comparative study was conducted to assess the contact and fumigant toxicities of eleven monoterpenes on two important stored products insects—, Sitophilus oryzae, the rice weevil, and Tribolium castaneum, the rust red flour beetle. The monoterpenes included: camphene, (+)-camphor, (−)-carvone, 1-8-cineole, cuminaldehyde, (l)-fenchone, geraniol, (−)-limonene, (−)-linalool, (−)-menthol, and myrcene. The inhibitory effect of these compounds on acetylcholinesterase (AChE) activity also was examined to explore their possible mode(s) of toxic action. Although most of the compounds were toxic to S. oryzae and T. castaneum, their toxicity varied with insect species and with the bioassay test. In contact toxicity assays, (−)-carvone, geraniol, and cuminaldehyde showed the highest toxicity against S. oryzae with LC₅₀ values of 28.17, 28.76, and 42.08 μg/cm², respectively. (−)-Carvone (LC₅₀ = 19.80 μg/cm²) was the most effective compound against T. castaneum, followed by cuminaldehyde (LC₅₀ = 32.59 μg/cm²). In contrast, camphene, (+)-camphor, 1-8-cineole, and myrcene had weak activity against both insects (i.e., LC₅₀ values above 500 μg/cm²). In fumigant toxicity assays, 1-8-cineole was the most effective against S. oryzae and T. castaneum (LC₅₀ = 14.19 and 17.16 mg/l, respectively). Structure-toxicity investigations revealed that (−)-carvone—, a ketone—, had the highest contact toxicity against the both insects. 1-8-Cineole—, an ether—, was the most potent fumigant against both insects. In vitro inhibition studies of AChE from adults of S. oryzae showed that cuminaldehyde most effectively inhibited enzyme activity at the two tested concentrations (0.01 and 0.05 M) followed by 1-8-cineole, (−)-limonene, and (l)-fenchone. 1-8-Cineole was the most potent inhibitor of AChE activity from T. castaneum larvae followed by (−)-carvone and (−)-limonene. The results of the present study indicate that (−)-carvone, 1,8-cineole, cuminaldehyde, (l)-fenchone, and (−)-limonene could be effective biocontrol agents against S. oryzae and T. castaneum.     

Bidirectional Selection for Novel Pheromone Blend Ratios in the Almond Moth, <Emphasis Type="Italic">Cadra cautella</Emphasis>

The sex pheromone of the almond moth, Cadra cautella, is a blend of (Z,E)-9,12-tetradecadienyl acetate (Z9,E12–14:Ac, the major component capable of inducing attraction alone) and (Z)-9-tetradecenyl acetate (Z9–14:Ac, the minor component, which is unattractive alone but augments attraction of the major component). In this study, the ratio of the two components responded to artificial directional selection in five of six selected lines, whereas no change was observed in the three control lines. The mean ratio (±SE) of Z9,E12–14:Ac to Z9–14:Ac went from 13.72 ± 1.02:1 to 20.13 ± 0.68:1 in high line 1, an increase of 47%. In the second high-selected line, the mean ratio (±SE) increased from 9.87 ± 0.54:1 to 15.89 ± 0.85:1, an increase of 61%. In low line 1, the mean ratio (±SE) in the parental generation was 10.74 ± 0.78:1 and 7.35 ± 0.41:1 in the last selected generation, a decrease of 32%. The response to selection was greater in low line 2, as the mean ratio (±SE) decreased from 10.11 ± 0.66:1 to 5.65 ± 0.55:1 in the last generation, a decrease of 44%. In low line 3, the mean ratio (±SE) in the parental generation was 13.63 ± 0.82:1 and 6.47 ± 0.26:1 in the last generation, a decrease of 53%. The response to selection was approximately symmetrical with a mean increase of 54% and a mean decrease of 43%. The increases in ratio observed in the high lines were caused by an increase in the titer of the Z9,E12–14:Ac component with no concurrent change in the titer of the component Z9–14:Ac. Among the low selected lines, the titers of both components increased; however, there was a greater relative increase in the titer of the component Z9–14:Ac. The absolute and relative titers of the sex pheromone components had decreased significantly in the F10 generation in some of the selected lines, five generations after the discontinuation of selection.     

Silicate and zirconium phosphate modified Nafion/PTFE composite membranes for high temperature PEMFC

The PEMFC performance of MEAs prepared from Nafion-212 (thickness 50 μm, Du Pont Co), porous poly(tetrafluoro ethylene) (PTFE, thickness 15 ~ 18 μm) film reinforced Nafion (NF, thickness 20 ± 2 μm), silicate hybridized NF (NF-Si, thickness 21 ± 2 μm), and zirconium phosphate hybridized NF (NF-Zr, thickness 21 ± 2 μm) membranes were investigated at 110 °C/ 51.7% RH, 120 °C/ 38.2% RH, and 130 °C/ 28.6% RH. We show PEMFC performances of these MEAs decrease in the sequence of: NF-Zr> NF-Si> NF> Nafion-212. The NF, NF-Si, and NF-Zr membranes have lower membrane thickness and lower Nafion content and require less water for proton transport than Nafion-212 at temperatures above 110 °C, and thus have higher conductivity and better PEMFC performance than Nafion-212. Incorporating silicate and zirconium phosphate into NF membranes enhances water retention of membranes at temperatures above 110 °C and improves PEMFC performances. Besides enhancing water retention, incorporating zirconium phosphate into membranes also provides more routes for proton transport via H⁺ exchange between H₃ ⁺O and HPO₄-Zr- and between H₂ ⁺PO₄-Zr- and HPO₄-Zr-. Thus NF-Zr has a higher conductivity and better PEMFC performance than NF and NF-Si.     

Polybenzimidazole/poly(tetrafluoro ethylene) composite membranes for high temperature proton exchange membrane fuel cells

A porous poly(tetrafluoro ethylene) (PTFE) thin film (thickness 16 ± 2 μm) is used as a supporting material for polybenzimidazole (PBI) to prepare the PBI/PTFE composite membrane (thickness 38 ± 2 μm). The perfluorosulfonic acid resin (Nafion) is used as a coupling agent at the interface between PTFE and PBI to improve the bonding between PBI and PTFE. The composite membrane, after doping with phosphoric acid, is used to prepare membrane electrode assemblies (MEAs). A 450 h continuous fuel cell life test at 160 °C with a fixed current density i = 200 mA cm⁻² and a 20 cycles cell on/off test, in which the fuel cell is operated at 160 °C with i = 200 mA cm⁻² for 12 h and then switched off at room temperature in an ambient environment for 12 h per cycle, are performed. Both tests show good fuel cell performances.     

Increasing DHA and EPA Concentrations Prolong Action Potential Durations and Reduce Transient Outward Potassium Currents in Rat Ventricular Myocytes

The goal of this study was to determine the mechanisms of n-3 polyunsaturated fatty acids (n-3 PUFA) on anti-arrhythmias and prevention of sudden death. The calcium-tolerant Sprague–Dawley rat ventricular myocytes were isolated by enzyme digestion. Effects of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on action potentials and transient outward potassium currents (I _to) of epicardial ventricular myocytes were investigated using whole-cell patch clamp techniques. Action potential durations (APDs) and I _to were observed in different concentrations of DHA and EPA. APD₂₅, APD₅₀, and APD₉₀ with 0.1 μmol/L DHA and EPA were prolonged less than 15% and 10%. However, APDs were prolonged in concentration-dependent manners when DHA and EPA were more than 1 μmol/L. APD₂₅, APD₅₀, and APD₉₀ were 7.7 ± 2.0, 21.2 ± 3.5, and 100.1 ± 9.8 ms respectively with 10 μmol/L DHA, and 7.2 ± 2.5, 12.8 ± 4.2, and 70.5 ± 10.7 ms respectively with 10 μmol/L EPA. I _to currents were gradually reduced with the increased concentrations of DHA and EPA from 1 to 100 μmol/L, and their half-inhibited concentrations were 2.3 ± 0.2 and 3.8 ± 0.6 μmol/L. The results showed APDs were prolonged and I _to current densities were gradually reduced with the increased concentrations of DHA and EPA. The anti-arrhythmia mechanisms of n-3 PUFA are complex, however, the effects of n-3 PUFA on action potentials and I _to may be one of the important mechanisms.     

Amphibian Chemical Defense: Antifungal Metabolites of the Microsymbiont <Emphasis Type="Italic">Janthinobacterium lividum</Emphasis> on the Salamander <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Disease has spurred declines in global amphibian populations. In particular, the fungal pathogen Batrachochytrium dendrobatidis has decimated amphibian diversity in some areas unaffected by habitat loss. However, there is little evidence to explain how some amphibian species persist despite infection or even clear the pathogen beyond detection. One hypothesis is that certain bacterial symbionts on the skin of amphibians inhibit the growth of the pathogen. An antifungal strain of Janthinobacterium lividum, isolated from the skin of the red-backed salamander Plethodon cinereus, produces antifungal metabolites at concentrations lethal to B. dendrobatidis. Antifungal metabolites were identified by using reversed phase high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance, and UV-Vis spectroscopy and tested for efficacy of inhibiting the pathogen. Two metabolites, indole-3-carboxaldehyde and violacein, inhibited the pathogen’s growth at relatively low concentrations (68.9 and 1.82 μM, respectively). Analysis of fresh salamander skin confirmed the presence of J. lividum and its metabolites on the skin of host salamanders in concentrations high enough to hinder or kill the pathogen (51 and 207 μM, respectively). These results support the hypothesis that cutaneous, mutualistic bacteria play a role in amphibian resistance to fungal disease. Exploitation of this biological process may provide long-term resistance to B. dendrobatidis for vulnerable amphibians and serve as a model for managing future emerging diseases in wildlife populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bioavailability and pharmacokinetics of bufadienolides‐loaded lipid microspheres after different administrations to rats

The degradation kinetics in vitro and pharmacokinetics of bufadienolides loaded in lipid microsphere (BU‐LM) after intravenous (IV) administration were investigated and compared with bufadienolides solution (BU‐S). The bioavailabilities of BU‐LM after intraportal (F_IP), intraduodenal (F_ID), and intragastric (F_IG) administrations to rats were also evaluated. The degradation kinetics showed that BU‐LM could protect bufadienolides from rapid metabolism in the plasma and gastrointestinal tract. The pharmacokinetic study revealed that BU‐LM after IV administration exhibited longer half‐life, longer mean residence time and higher apparent volume of distribution than intravenous BU‐S. After different routes of administration of BU‐LM, cinobufagin (C), and resibufogenin (R) were metabolized completely and could not be detected at an oral dose of 6 mg/kg. Furthermore, C exhibited a complete hepatic first‐pass effect after IP administration of 0.5 mg/kg BU‐LM. Bufalin (B), the lowest component of the BU‐LM could be detected at any administration and the F_IG, F_ID, and F_IP for B were 43.05, 88.93, and 93.31%, respectively. R had a F_ID and F_IP of only 1.51 and 2.77%, respectively. The results demonstrated that the stomach appeared to be the dominating site of metabolism for B and the liver for C and R. Practical applications: Bufadienolides, consisting of bufalin (B), binobufagin (C), and resibufogenin (R) are easily hydrolyzed (catalyzed by acids, bases, and enzymes) and are metabolized rapidly in rats because of their chemical structures, especially C. However, the three bufadienolides could be protected from degradation effectively and the degradation half‐lives could be extended to different degrees by using lipid microspheres (LM) as a delivery system. Also, after intravenous administration to rats, bufadienolides‐loaded lipid microspheres (BU‐LM) could slow down the metabolism and elimination of bufadienolides, delay the release and increase the targeted effect of the drug compared with bufadienolides aqueous solution (BU‐S_iv). Consequently, LM is a promising alternative vehicle for bufadienolides.     

Differential Attractiveness of Potato Tuber Volatiles to <Emphasis Type="Italic">Phthorimaea operculella</Emphasis> (Gelechiidae) and the Predator <Emphasis Type="Italic">Orius insidiosus</Emphasis> (Anthocoridae)

The behavioral responses of the potato tuberworm moth Phthorimaea operculella and the polyphagous predator Orius insidiosus to volatiles emanating from exposed tubers were studied by four-arm olfactometer bioassays. Mated females of P. operculella distinguished volatiles released by intact potato tubers from volatiles damaged mechanically or by conspecific larvae. Volatiles from intact potato tubers were attractive to them. On the other hand, unmated females of P. operculella did not respond to tuber volatiles. Adults of O. insidiosus were attracted to volatiles from tubers damaged by P. operculella larvae, but did not respond to intact or mechanically damaged tubers. Methyl jasmonate (MeJA) was the only compound identified from the headspace of potato tubers (GC-MS of direct headspace sampling). The amount varied with the type of induction, being 0.001 ± 0.0003 ng g⁻¹ in tissues of intact fresh tubers, 0.002 ± 0.0007 ng g⁻¹ in mechanically damaged tubers, and showing a six- to tenfold increase in P. operculella damaged tubers (0.090 ± 0.006 ng g⁻¹). Behavioral bioassays with synthetic MeJA confirmed that the response of the insects is dependent on MeJA concentration. Mated females of P. operculella showed the highest response at 0.001 ng g⁻¹ (concentration released by intact tubers), whereas O. insidiosus showed the highest response, between 0.01 and 0.05 ng g⁻¹, which is close to the concentration released by P. operculella damaged tubers. Based on these results, we postulate that P. operculella and O. insidiosus have adapted their responses to plant volatiles differently, enabling them to locate suitable hosts or prey.     

Biparental Endowment of Endogenous Defensive Alkaloids in <Emphasis Type="Italic">Epilachna paenulata</Emphasis>

Coccinellid beetles contain a variety of defensive alkaloids that render them unpalatable to predators. Epilachna paenulata (Coleoptera: Coccinellidae) is a South American ladybird beetle that feeds on plants of the Cucurbitaceae family. The defensive chemistry of E. paenulata has been characterized as a mixture of systemic piperidine, homotropane, and pyrrolidine alkaloids. Whole body extracts of adult beetles contain four major alkaloids: 2-(2′-oxopropyl)-6-methylpiperidine (1); 1-(6-methyl-2,3,4,5-tetrahydro-pyridin-2-yl)-propan-2-one (2); 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3); and 1-(2″-hydroxyethyl)-2-(12′-aminotridecyl)-pyrrolidine (4). Comparative studies of the defensive chemistry of eggs, larvae, pupae, and adults showed differences in alkaloid composition and concentration among life stages. While adults contained mainly the homotropane 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3), eggs showed the highest concentration of the piperidine 2-(2′-oxopropyl)-6-methylpiperidine (1). We studied the origin of this alkaloid in the eggs by feeding newly emerged, virgin adult beetles with [2-¹³C]-labeled acetate, and by performing crosses between ¹³C-fed and unlabeled males and females. GC-MS analysis of alkaloids from ¹³C-fed males and females showed high incorporation of ¹³C into the alkaloids, as evidenced from a 20–30% increase of isotopic peaks in diagnostic fragment ions, confirming the expected endogenous origin of these alkaloids. In addition, analyses of eggs from different crosses showed that labeled alkaloids from both parents are incorporated into eggs, indicating that E. paenulata males transfer alkaloids to the females at mating. Biparental endowment of chemical defenses into eggs has been shown previously in insects that acquire defensive compounds from dietary sources. To our knowledge, this is the first report of biparental egg endowment of endogenous defenses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of an Efficient Pheromone-Based Trapping Method for the Banana Root Borer <Emphasis Type="Italic">Cosmopolites sordidus</Emphasis>

The banana root borer Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is a major pest of bananas throughout the world. Chemical control is both undesirable and expensive, where biological control alternatives are limited, and pheromone-based trapping results in low captures. In this study, several important factors that affect pheromone-based catches, such as trap type, trap dimensions, and color and position of the traps, were optimized. Ground traps were found to be superior to ramp and pitfall traps, and larger traps (40 × 25 cm and above) were more efficient than smaller ones (30 × 15 cm). In a color-choice test, the banana weevil clearly preferred brown traps over yellow, red, gray, blue, black, white, and green, with mahogany being more attractive than other shades of brown. In addition, pheromone baited ground traps positioned in the shade of the canopy caught significantly more adults than those placed in sunlight. Therefore, mahogany-brown ground traps 40 × 25 cm appear to be the most efficient at catching C. sordidus adults and have the greatest potential for use in mass trapping and programs for eradication of this pest.     

A Male-produced Aggregation Pheromone Blend Consisting of Alkanediols,Terpenoids, and an Aromatic Alcohol from the Cerambycid Beetle <Emphasis Type="Italic">Megacyllene caryae</Emphasis>

Bioassays conducted with a Y-tube olfactometer provided evidence that both sexes of the cerambycid beetle Megacyllene caryae (Gahan) were attracted to odor produced by males. Odor collected from male M. caryae contained eight male-specific compounds: a 10:1 blend of (2S,3R)- and (2R,3S)-2,3-hexanediols (representing 3.2 ± 1.3% of the total male-specific compounds), (S)-(−)-limonene (3.1 ± 1.7%), 2-phenylethanol (8.0 ± 2.4%), (−)-α-terpineol (10.0 ± 2.8%), nerol (2.1 ± 1.5%), neral (63.3 ± 7.3%), and geranial (8.8 ± 2.4%). Initial field bioassays determined that none of these compounds was attractive as a single component. Further field trials that used a subtractive bioassay strategy determined that both sexes were attracted to the complete blend of synthetic components, but the elimination of any one component resulted in a decline in trap captures. Blends that were missing (2S,3R)-2,3-hexanediol, (2R,3S)-2,3-hexanediol, or citral (a 1:1 mixture of neral and geranial) attracted no more beetles than did controls. A pheromone blend of this complexity, composed of alkanediols, terpenoids, and aromatic alcohols, is unprecedented for cerambycid species.     

Sequestration of Furostanol Saponins by <Emphasis Type="Italic">Monophadnus</Emphasis> Sawfly Larvae

Sawfly larvae of the tribe Phymatocerini (Hymenoptera: Tenthredinidae), which are specialized on toxic plants in the orders Liliales and Ranunculales, exude a droplet of deterrent hemolymph upon attack by a predator. We investigated whether secondary plant metabolites from Ranunculaceae leaves are sequestered by phymatocerine Monophadnus species, i.e., Monophadnus alpicola feeding upon Pulsatilla alpina and Monophadnus monticola feeding upon Ranunculus lanuginosus. Moreover, two undescribed Monophadnus species were studied: species A collected from Helleborus foetidus and species B collected from Helleborus viridis. Comparative high-performance liquid chromatographic–photodiode array detection–electrospray ionization–mass spectrometric analyses of plant leaf and insect hemolymph extracts revealed the presence of furostanol saponins in all samples. Larvae of species A and B actively sequestered (25R)-26-[(α-l-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl O-β-d-glucopyranosyl-(1→3)-O-[6-acetyl-β-d-glucopyranosyl-(1→3)]-O-β-d-glucopyranoside (compound 1). This compound occurred at a 65- to 200-fold higher concentration in the hemolymph of the two species (1.6 and 17.5 μmol/g FW, respectively) than in their host plant (0.008 and 0.268 μmol/g FW, respectively). In M. monticola, compound 1 was found at a concentration (1.2 μmol/g FW) similar to that in the host plant (1.36 μmol/g FW). The compound could not be detected consistently in M. alpicola larvae where, however, a related saponin may be present. Additional furostanol saponins were found in H. foetidus and H. viridis, but not in the two Monophadnus species feeding on them, indicating that sequestration of compound 1 is a highly specific process. In laboratory bioassays, crude hemolymph of three Monophadnus species showed a significant feeding deterrent activity against a potential predator, Myrmica rubra ant workers. Isolated furostanol saponins were also active against the ants, at a concentration range similar to that found in the hemolymph. Thus, these compounds seem to play a major role for chemical defense of Monophadnus larvae, although other plant secondary metabolites (glycosylated ecdysteroids) were also detected in their hemolymph. Physiological and ecological implications of the sequestered furostanol saponins are discussed. Dedicated to the memory of Professor Ivano Morelli (1940–2005)     

The Chlorophyll Catabolite,Pheophorbide <Emphasis Type="Italic">a</Emphasis>, Confers Predation Resistance in a Larval Tortoise Beetle Shield Defense

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 μg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 μg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 μg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 μg added Pha were comparable to intact shields, even though the Pha concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.     

Aggregation Pheromone of the Qinghai Spruce Bark Beetle, <Emphasis Type="Italic">Ips nitidus</Emphasis> Eggers

Volatiles from hindgut extracts of males of the Qinghai spruce bark beetle, Ips nitidus, from different attack phases (phase 1: unpaired males and phases 2–4: males joined with one to three females) and hindgut extracts of mated females were analyzed by gas chromatography–mass spectrometry (GC–MS)/flame ionization detection (FID) with both polar and enantioselective columns. The GC–MS/FID analyses demonstrated that unpaired males from attack phase 1 (nuptial chamber constructed) produced 2-methyl-3-buten-2-ol, approx. 74%-(−)-ipsdienol, and (−)-cis-verbenol as major hindgut components, and (−)-trans-verbenol, (−)-ipsenol, (−)-verbenone, myrtenol, and 2-phenylethanol as minor or trace components. The quantities of 2-methyl-3-buten-2-ol and especially ipsdienol decreased after mating during phases 2–4, whereas the quantities of (−)-cis- and (−)-trans-verbenol did not change. In contrast, the quantity of (−)-ipsenol seemed to increase as mating activity progressed. After mating with three females (harem size = 3; phase 4), only trace to small amounts of male-specific compounds were detected from I. nitidus male hindguts. Chemical analysis of the hindgut extracts of mated females showed only trace amounts of semiochemicals. A field-trapping bioassay in Qinghai, China showed that the four-component “full blend” containing the three major components, 2-methyl-3-buten-2-ol, (±)-ipsdienol, and (−)-cis-verbenol, plus a minor component, (−)-trans-verbenol, caught significantly more I. nitidus (♂/♀ = 1:2.2) than did the unbaited control and two binary blends. The replacement of (±)-ipsdienol with nearly enantiomerically pure (−)-ipsdienol in the “full blend” significantly reduced trap catches, which suggests that both enantiomers are needed for attraction. On the other hand, removal of (−)-trans-verbenol from the active “full blend” had no significant effect on trap catches. Our results suggest that the three major components, 2-methyl-3-buten-2-ol, 74%-(−)-ipsdienol, and (−)-cis-verbenol (at 7:2:1), produced by unpaired fed males, are likely the aggregation pheromone components of I. nitidus, thus representing the first characterization of an aggregation pheromone system of a bark beetle native solely to China.     

Reaction of MoSi<Subscript>2</Subscript> with niobium and tantalum diborides

Results are provided for a study of the reaction of MoSi₂ with niobium and tantalum diborides. It is established that sections of MoSi₂–Nb(Ta)B₂ within the quaternary system Mo–Si–B–Nb(Ta) are described by eutectic composition diagrams with a eutectic temperature equal to 1870 and (1890 ± 30)°C. The diboride phase content is 20 and 14 mol.% respectively in systems with NbB₂ and TaB₂.     

Fatty acid composition of brain glycerophospholipids in peroxisomal disorders

This paper shows for the first time the differential fatty acid composition of ethanolamine plasmalogens (EP) and phosphatidylethanolamine (PE) in the brains of 12 patients with disorders of peroxisomal biogenesis and compares the results to normal values for the age. Other important glycerophospholipids (GPL), such as phosphatidylserine (PS) and phosphatidylcholine (PC), are also included in this study. GPL were separated by two-dimensional thin-layer chromatography, and their fatty acid composition was determined by capillary column gas-liquid chromatography. Total brain GPL were slightly decreased in peroxisomal disorders (27.98±2.95 μmol/g in the patients against 34.5±6.21 μmol/g in age-matched controls, P=0.005), and the distribution of the different GPL classes was much altered. In confirmation of known data, EP were very much decreased (2.18±1.3 μmol/g in the patients against 6.9±2.3 μmol/g in controls) at the expense of PE, which was increased (8.58±2.17 μmol/g in the patients against 5.97±0.58 μmol/g in controls, P<0.005). PS and PC were both significantly decreased (P=0.0001 and P=0.037, respectively). The polyunsaturated fatty acid (PUFA) composition of all the GPL fractions was markedly abnormal. In absolute terms, docosahexaenoic acid (22∶6n−3) was drastically decreased in all GPL classes (always at the P<0.0001 level) while arachidonic acid (20∶4n−6) was increased in PE and PS (P<0.001 in both cases). In the alkenyl acyl form, EP, 22∶6n−3, and 20∶4n−6 were both very significantly decreased (P<0.0001), although the former was always the most affected. The myelin PUFA adrenic acid (22∶4n−6) was decreased in EP (P<0.0001) and slightly increased in PS (P<0.05). The changes found confirm that 22∶6n−3 deficiency is a predominant defect in the brain in peroxisomal disorders.