Development of Tolerance to the Dietary Plant Secondary Metabolite 1,8-cineole by the Brushtail Possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The common brushtail possum (Trichosurus vulpecula) is a generalist herbivore whose diet includes Eucalyptus leaves that are well defended by plant secondary metabolites (PSM) such as the terpene 1,8-cineole (cineole). We accustomed possums to a terpene-free diet, then challenged them with the addition of 2% cineole to the diet. Initially, there was a 50% reduction in total overnight food consumption associated with a marked decrease in the mass of the major feeding bout. After nine nights, however, cineole tolerance had developed as total food consumption had returned to the control amount. Compared to the control diet, the cineole diet was eaten in a larger number of smaller bouts, which were also eaten at a slower rate. The experiment was repeated with animals that had been accustomed to day-time feeding to take blood samples during feeding sessions. Feeding variables and blood concentration data for cineole were compared on the first and seventh day of the cineole diet. Although the total food consumed increased 2.5-fold after 7 days of the cineole diet, there was no increase in average blood cineole concentration, measured as the area under the concentration–time curve. This indicates that induction of liver enzymes resulted in greater pre-systemic metabolism of cineole and reduced bioavailability. The maximum tolerated blood concentration of cineole also increased, suggesting some adaptation of the central nervous system to the cineole aversive effects. This appears to be the first report in a vertebrate herbivore that consumption of a dietary PSM leads to metabolism induction and that this contributes to development of tolerance to the PSM. Overall, herbivores adapt to newly encountered dietary PSMs by immediate changes in feeding behavior followed by development of increased metabolism of PSM and probably diminished cellular responsiveness to effects.     

Laboratory Evaluation of Odor Preferences of the Brushtail Possum

The Australian brushtail possum (Trichosurus vulpecula) is the major vertebrate pest in New Zealand. Possums cause significant damage to the country's native and exotic forests and, as a vector of bovine tuberculosis, are a serious threat to the country's meat industry. Strong smelling odors are often used as lures during possum control operations in New Zealand, but little is known about the preferences of possums for these odurs. A test was designed to determine the preferences of captive wild possums for a number of odors commonly used as lures. To assess the effectiveness of the method, the possums were tested for their responses to a familiar food odor (apple) versus no odor. The possums spent significantly more time investigating the apple odor and also sniffed it more often than the no-odor control in the 15-min test period. The same procedure, however, showed that five odors commonly used as lures (almond, cloves, cinnamon, peanut, and orange) were no more preferable than water. Our results suggest that these unfamiliar odors are either equally attractive or unattractive to possums and that odors that are more familiar to these animals may be more appropriate as lures.     

Role of Detoxification of Plant Secondary Compounds on Diet Breadth in a Mammalian Herbivore, Trichosurus vulpecula

Theory predicts that mammalian herbivores detoxify different classes of plant secondary compounds via separate metabolic pathways and that generalist herbivores maintain broad diet breadth to avoid overloading individual detoxification pathways. We tested the hypothesis that a generalist marsupial herbivore, the common brushtail possum, Trichosurus vulpecula, can maintain a higher intake of food when allowed to select from two diets containing different profiles of secondary compounds (phenolics and terpenes) than when given access to the diets individually. Diets consisted of a fruit and vegetable mash to which was added ground leaves of either Eucalyptus melliodora or E. radiata. E. melliodora and E. radiata differ in their concentrations and types of secondary compounds. Brushtail possums include these eucalypt species as part of their natural diet. We measured food consumption and detoxification metabolites of possums on these diets. Consistent with the hypothesis, animals presented with a choice of both diets consumed more food than animals given diets singly. One of the two indicators of detoxification, acid load in urine, differed significantly between diets while the other, glucuronic acid, did not. These results provide partial support for the hypothesis that diet breadth is governed by detoxification abilities.     

Triacylglycerol Estolides,a New Class of Mammalian Lipids,in the Paracloacal Gland of the Brushtail Possum (Trichosurus vulpecula)

The paracloacal glands are the most prevalent scent glands in marsupials, and previous investigation of their secretions in the brushtail possum (Trichosurus vulpecula) has identified many odorous compounds together with large amounts of neutral lipids. We have examined the lipids by LC–MS, generating ammonium adducts of acylglycerols by electrospray ionisation. Chromatograms showed a complex mixture of coeluting acylglycerols, with m/z from about 404 to 1048. Plots of single [M + NH₄]⁺ ions showed three groups of lipids clearly separated by retention time. MS–MS enabled triacylglycerols and diacylglycerol ethers to be identified from neutral losses and formation of diacylglycerols and other product ions. The earliest‐eluting lipids were found to be triacylglycerol estolides, in which a fourth fatty acid forms an ester link with a hydroxy fatty acid attached to the glycerol chain. This is the first report of triacylglycerol estolides in animals. They form a complex mixture with the triacylglycerols and diacylglycerol ethers of lipids with short‐ and long‐chain fatty acids with varying degrees of unsaturation. This complexity suggests a functional role, possibly in social communication.     

Rapid Absorption of Dietary 1,8-Cineole Results in Critical Blood Concentration of Cineole and Immediate Cessation of Eating in the Common Brushtail Possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The blood concentration of 1,8-cineole and its metabolites was measured in six male brushtail possums while they voluntarily fed on diets laced with varying concentrations of cineole for 3 d. On the third day, blood samples were collected during and after each bout of feeding for 3 hr. Blood cineole was measured by using headspace solid-phase microextraction (SPME), while cineole metabolites were measured by liquid–liquid extraction followed by gas chromatography-mass spectroscopy. Feeding patterns were measured by continual recording of residual food weight and time. Cineole absorption was rapid, resulting in a peak blood concentration at the end of each feeding bout. The blood concentration of cineole did not exceed a critical value (51.8 ± 14.1 μmol/l) regardless of the concentration in the diet. Food and, therefore, cineole intake was regulated. The amount of food ingested in the first feeding bout decreased from 236 ± 52 g on the control diet to 36 ± 20 g on the 4% cineole diet. The amount of cineole ingested in the first bout (1.18 ± 1.10 g) was the same regardless of the dietary concentration and was controlled by the size of the meal. Total food eaten during the 7-hr feeding session decreased by 64% from 368 ± 94 g (control diet) to 131 ± 52 g (4% diet). Total cineole intake increased from 2.47 ± 0.60 g (1% diet) to 5.05 ± 2.41 g (4% diet). Cineole metabolites accumulated throughout the sampling period and were generally still rising at the end of blood sampling period. Blood levels of metabolites were at least 10-fold higher than cineole levels. The immediate control of feeding seems to be regulated by blood levels of cineole, whereas metabolites are likely to be more important in regulating the chronic ingestion of cineole.     

Analysis of secretions from scent-producing glands of brushtail possum (Trichosorus vulpecula Kerr)

A characterization at a molecular level of the chemical composition of the secretions of the two pairs of paracloacal glands and of the sternal sebum of the brushtail possum (Trichosorus vulpecula Kerr) has been undertaken with a view to evaluating the potential of volatile pheromone components as species-specific attractants for use in novel baiting systems. Particular attention has been given to the respective fatty acid fractions produced by chemical hydrolysis, since these are believed to be the products of postemission microbial degradation (fermentation) of the secretions. In all instances, the highly complex distribution of the constituents present in these organic components of the secretions were shown to be virtually identical in adult males and females. A unique suite of low-molecular-weight branched-chain carboxylic acids has been shown to be produced by chemical degradation of the holocrine (oil-secreting) gland secretion. This odor signature is suggested to function as a unique scenting-the-habitat pheromone that might act as an attractant to all members of the species.     

Problems Encountered in Feeding Microencapsulated Oils to a Folivorous Marsupial, the Common Ringtail Possum (Pseudocheirus peregrinus)

In our study of interactions between marsupial folivores and Eucalyptus, we have examined the role of terpenes, including the use of bioassay experiments where terpenes are added to an artificial diet. In pursuit of greater ecological realism in these experiments, we have sought means of packaging terpenes more realistically, by using microencapsulation to simulate oil glands of leaves. We report here on a preliminary experiment with microencapsulated olive oil (intended for use as an experimental control) in which the food intake of the animals was substantially reduced, to the point that starvation appeared imminent and we aborted the experiment. We discuss why this occurred and recommend caution to others intending to use microencapsulated terpenes (or other oils) in herbivore diets.     

Effects of two plant secondary metabolites,cineole and gallic acid,on nightly feeding patterns of the common brushtail possum

We investigated effects of two plant secondary metabolites (PSMs), cineole and gallic acid, on the nightly feeding behavior of the common brushtail possum (Trichosurus vulpecula), a generalist folivore. We tested whether possums altered their feeding behavior in response to increasing levels of cineole, a dietary terpene. Possums were fed artificial diets containing three levels of cineole; zero (basal diet), medium (6.8% of total dry matter, DM), and high (15.3% DM). In another experiment, we introduced gallic acid, a dietary phenolic, into the diets. Possums were offered a Choice PSM diet (cineole and gallic acid diets simultaneously) or a No-Choice PSM diet (containing either cineole or gallic acid). Detoxification products of cineole and gallic acid were examined in urine to determine that different detoxification pathways were utilized in the elimination of each compound. With increasing cineole levels, possums ate less, had smaller feeding bouts, and had a lower rate of intake, but did not extend their total nightly feeding time. Possums offered the Choice PSM diet, compared with the No-Choice diets, ate more, had larger feeding bouts, and tended to increase their rate of intake. Results from the urinary analysis indicated that gallic acid and cineole were not involved in competing detoxification pathways in brushtail possums. There was also a significant sex effect: females ate more overall, ate more per feeding bout, and ate at a higher rate than males. These results indicate that PSMs not only constrain overall intake, but that possums alter their feeding behavior in response to them. Altered feeding patterns may reduce the negative influence of PSMs on intake.     

Inheritance Of Resistance to Mammalian Herbivores and of Plant Defensive Chemistry in an <Emphasis Type="Italic">Eucalyptus</Emphasis> Species

Hybridization in plants provides an opportunity to investigate the patterns of inheritance of hybrid resistance to herbivores, and of the plant mechanisms conferring this resistance such as plant secondary metabolites. We investigated how inter-race differences in resistance of Eucalyptus globulus to a generalist mammalian herbivore, Trichosurus vulpecula, are inherited in their F₁ hybrids. We assessed browsing damage of 3-year-old trees in a common environment field trial on four hybrid types of known progeny. The progeny were artificial intra-race crosses and reciprocal inter-race F₁ hybrids of two geographically distinct populations (races) of E. globulus north-eastern Tasmania and south-eastern Tasmania. Populations of trees from north-eastern Tasmania are relatively susceptible to browsing by T. vulpecula, while populations from south-eastern Tasmania are more resistant. We assessed the preferences of these trees in a series of paired feeding trials with captive animals to test the field trial results and also investigated the patterns of inheritance of plant secondary metabolites. Our results demonstrated that the phenotypic expression of resistance of the inter-race F₁ hybrids supported the additive pattern of inheritance, as these hybrids were intermediate in resistance compared to the pure parental hybrids. The expression of plant secondary metabolites in the F₁ hybrids varied among major groups of individual compounds. The most common pattern supported was dominance towards one of the parental types. Together, condensed tannins and essential oils appeared to explain the observed patterns of resistance among the four hybrid types. While both chemical groups were inherited in a dominant manner in the inter-race F₁ hybrids, the direction of dominance was opposite. Their combined concentration, however, was inherited in an additive manner, consistent with the phenotypic differences in browsing.     

Comparative Metabolism of Dietary Terpene, p-Cymene, in Generalist and Specialist Folivorous Marsupials

The urinary metabolites of the monoterpene, p-cymene, were studied in three marsupial species: a generalist herbivore, the brushtail possum (Trichosurus vulpecula), and two specialist folivores, the greater glider (Petauroides volans) and the ringtail possum (Pseudocheirus peregrinus), as well as in the laboratory rat (Rattus norvegicus). Each animal was dosed, intragastrically, with single doses of p-cymene (0.37 mmol/kg and/or 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods. Quantitative analysis of urinary metabolites by extraction, gas chromatography, and mass spectrometry gave a mean recovery of 64% (range 52–74%) of the administered dose in 48 hr for the four species. No fecal metabolites were found. A species-specific pattern of metabolite excretion was evident and reflected the natural occurrence of p-cymene (and terpenes in general) in the diet. If the metabolites excreted are grouped according to the total number of oxygen atoms added (one to four), then the rat excreted metabolites encompassing all degrees of oxidation, but predominantly a monooxygenated metabolite. The brushtail possum excreted metabolites having two, three, and four oxygen atoms added. The ringtail possum and greater glider only excreted metabolites with three or four oxygen atoms. Conjugation played a significant role in the excretion of oxidized metabolites in only the brushtail possums and the rat. These findings indicate that species encountering terpenes, such as p-cymene, in their normal diet have developed efficient oxidation pathways to eliminate them. This oxidative efficiency could also reduce the necessity for subsequent conjugation of metabolites which minimizes further demands on a nutritionally limited diet.     

Comparative Detoxification of Plant (Magnolia virginiana) Allelochemicals by Generalist and Specialist Saturniid Silkmoths

The foliage of sweetbay magnolia (Magnolia virginana) contains at least two biologically active phenylpropanoid compounds (magnolol and a biphenyl ether) that are toxic to a number of generalist insect herbivores. These compounds have little effect on caterpillars of the sweetbay silkmoth, C. securifera, which is a specialist on sweetbay, but they are toxic to two closely related silkmoths, C. angulifera and C. promethea. To understand the influence of phytochemistry on the evolution of host use and feeding specialization in Callosamia, the detoxification capability of C. securifera was compared with that of C. angulifera and C. promethea. Degradation of magnolol and the biphenyl ether by midgut homogenate of the sweetbay specialist was NADPH-dependent and inhibited by piperonyl butoxide, suggesting the involvement of cytochrome P-450 detoxification enzymes. Both were degraded three times faster in the specialist compared to the unadapted herbivores. Higher rates of degradation could not be induced in the polyphagous C. promethea by a mixture of magnolol and the biphenyl ether or by the P-450 inducer pentamethylbenzene, nor did activity vary significantly when larvae were reared on different host plants. Use of sweetbay by Callosamia silkmoths appears to be dependent on their ability to degrade host toxins rapidly via midgut detoxification enzymes. Moreover, the intraspecific comparisons contradict the common prediction that higher levels of cytochrome P-450 activity are found in more polyphagous species; instead, P-450 activity is more closely associated with specific chemical attributes of the herbivores' host plants.     

Evaluation of Synergism in the Feeding Deterrence of Some Furanocoumarins on Spodoptera littoralis

The phagodepression activity of five furanocoumarins (FC), bergapten, xanthotoxin, psoralen, imperatorin, and angelicin, has been studied against larvae of Egyptian cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) using a leaf-disk choice bioassay. The dose range used was 0–10 g/cm² for linear furocoumarins and 0–30 g/cm² for angelicin, the angular furanocoumarin. Dose–feeding deterrency activity correlations were governed by various sigmoidal functions, except in the case of imperatorin and bergapten, which had dose–response curves showing irregular traces with two maxima. All five FCs induced various degrees of phagodepression in S. littoralis; at 3 g/cm², the relative feeding deterrence was bergapten = xanthotoxin > psoralen = imperatorin = angelicin. Structure–activity correlations indicated that a methoxy group on C-5 or C-8 enhanced the activity. Comparison of experimental feeding deterrence of binary mixtures of imperatorin with xanthotoxin, bergapten, or psoralen at various relative concentrations with the calculated additive activity of each combination indicated that a proportion of 40% or more of imperatorin may exert a greater antifeedant effect on S. littoralis than the sum of individual compounds. The effect of angelicin also was examined in combination with psoralen, both of which are present in Psoralea plants. Their mixtures yielded a clear synergistic effect in the 20–80% angelicin range (coactivity coefficient = 124–133). By contrast, the effect of angelicin on xanthotoxin was only additive. In the dose–response curves of imperatorin/bergapten combinations, synergism was found at >60% imperatorin relative concentration and 1 g/cm², whereas lower proportions led to antagonism. The threshold between the two opposing effects was found to depend on the total FC concentration employed. Some natural systems contain FCs in the range of synergistic proportions recorded here and, thus, may have been produced by the host to increase its defensive effect at a lower metabolic cost.     

Metabolism of 1,8-cineole in tea tree (Melaleuca alternifolia andM. linariifolia) by pyrgo beetle (Paropsisterna tigrina)

The frass of the pyrgo beetle (Paropsisterna tigrina) feeding on commercial plantations of the terpinen-4-ol chemical variety of the Australian tea tree.Melaleuca alternifolia, was found to contain a volatile oil almost identical to the essential oil of the ingested leaf. When beetles were fed leaf containing substantial quantities of 1,8-cineole, the predominant frass metabolite as determined by MS, IR,¹³C and¹H NMR, GC, and CoGC was (+)-2-hydroxycineole. Both male and female adults and larvae metabolizedMelaleuca oils in similar ways.     

Behavioral (Feeding) Responses of the Crayfish, Procambarus clarkii, to Natural Dietary Items and Common Components of Formulated Crustacean Feeds

We have examined the behavioral (feeding) response of Procambarus clarkii to natural dietary items (zooplankton, live fishes, dead fishes, and fish eggs) and common components of formulated feeds used in the aquaculture industry (soybean meal, fish meal, corn meal, alfalfa meal, and vitamin C). The feeding response by P. clarkii was determined using an ordinally ranked, whole-animal bioassay that included the following behaviors: (1) movement of the maxillipeds for longer than three seconds, (2) increased movement of the walking legs with dactyl probing, (3) movement of walking legs to the mouth, and (4) orientation of the entire body towards the odor source. Feeding behavior was determined in response to intact items, bathwater containing aqueous leachates from intact items, water and methanol fractions of bathwater eluted through a C₁₈ resin flash chromatography column, and size fractions of bathwater containing either molecules 10,000 Da or molecules >10,000 Da. All fractions tested were significantly stimulatory. Zooplankton was the most stimulatory of the natural dietary items tested. However, the C₁₈ water fraction of the soybean meal bathwater before size fractionation (containing molecules both <10,000 and >10,000 Da) was the most stimulatory of the common feed components and more stimulatory than the zooplankton. Proximate analysis indicated that the compounds present in this fraction were ca. 51% soluble carbohydrate, 4% soluble protein, and 45% unknown (assumed to be insoluble carbohydrates, insoluble proteins, and ash). We hypothesize that the primary compounds in soybean meal responsible for eliciting a feeding response in P. clarkii are soluble carbohydrates and/or glycoproteins.     

Identification of a four-component sex pheromone of the female oriental fruit moth,Grapholitha molesta (Lepidoptera: Tortricidae)

The female-emitted pheromone ofGrapholitha molesta (Busck), the Oriental fruit moth, was collected by holding females in glass flasks during calling. Flask washes were found to contain four pheromone components: (Z)-8-dodecenyl acetate and (E)-8-dodecenyl acetate in a 1007 ratio, and (Z)-8-dodecen-1-ol and dodecanol in a 10020 ratio. The ratio of (Z)-8-acetate to (Z)-8-dodecen-1-ol was approx. 10030. Approximately 0.1–0.2 ng of pheromone was recovered per female per hour of calling.Published as Journal Article No. 8598 of the Michigan State University Agricultural Experiment Station.     

Sex pheromone of the armyworm,Pseudaletia unipuncta (Haworth) (Lepidoptera: Noctuidae)

(Z)-11-Hexadecen-1-ol acetate (Z11–16Ac) free of theE isomer (<1%), hexadecan-1-ol acetate (16 Ac), and a hexadecen-1-ol [the (Z)-11 isomer based on the retention time on a Carbowax capillary column] were identified in extracts of the sex pheromone glands of adult virgin female armyworms,Pseudaletia unipuncta. Also, gas Chromatographic retention times on polar and nonpolar columns indicated the possible presence of (Z)-9-hexadecen-1-ol acetate (Z9–16Ac). The ratioZ11–16Ac/16Ac/Z11–16OH/Z9–16Ac was 10.150.130.02. Infield testsZ11–16 Ac was attractive alone, and the addition ofZ9–16Ac,Zll–16 OH, or 16 Ac singly or in combination in ratios found in the gland did not increase trap capture.Mention of a proprietary product does not constitute an endorsement of this product by the USDA.     

Feeding responses of snowshoe hares (Lepus americanus) to volatile constituents of red fox (Vulpes vulpes) urine

This study investigated the influence of the volatile constituents of red fox (Vulpes vulpes) urine in suppressing feeding by snowshoe hares (Lepus americanus) on coniferous tree seedlings. Pen and field bioassays indicated that the odor of fox urine and its principal component, 3-methyl-3-butenyl methyl sulfide, had a negative effect on feeding behavior of hares. The other sulfur-containing compounds, 2-phenylethyl methyl sulfide and 3-methylbutyl methyl sulfide, as well as six other constituents, were not effective. Synthetic urine mixtures composed of eight and nine volatile constituents, respectively, did not suppress feeding in pen bioassays. However, the mixture of eight compounds in a field bioassay did result in significantly fewer seedlings being eaten by hares than in the control. 3-Methyl-3-butenyl methyl sulfide may act as an interspecific chemical signal which induces a fear or avoidance response in hares. Additional work is required to determine the optimum concentration and release system for 3-methyl-3-butenyl methyl sulfide to be used as a mammalian semiochemical in crop protection.     

Chemical suppression of feeding in larval weakfish (Cynoscion regalis) by trochophores of the serpulid polychaeteHydroides dianthus

We studied the effect of exudates from trochophore larvae of the polychaeteHydroides dianthus on feeding in larval weakfish (Cynoscion regalis). Laboratory prey consisted ofH. dianthus trochophores and/or comparably sized rotifers (Brachionus plicatilis). When experiments were conducted in filtered seawater, ingestion of rotifers was always greater than ingestion of trochophores. However, consumption of rotifers was depressed when water fromH. dianthus cultures (=trochophore water) was the experimental medium. The same effect was noted whether we added trochophore water from polychaete cultures that were two or five days postfertilization. However, no effect was noted when we used water from rotifer cultures. We concluded thatH. dianthus trochophores release a water-soluble compound that inhibits feeding in weakfish larvae.     

Identification of volatile sex pheromone components released by the southern armyworm,Spodoptera eridania (Cramer)

Analysis of sex pheromone gland extracts and volatile pheromone components collected from the calling female southern armyworm,Spodoptera eridania (Cramer), by high-resolution capillary gas chromatography and mass spectroscopy indicated that a number of 14-carbon mono- and diunsaturated acetates and a monounsaturated 16-carbon acetate were produced. Gland extracts also indicated the presence of (Z)-9-tetradecen-1-ol. However, this compound was not found in collections of volatiles. Field trapping studies indicated that the volatile blend composed of (Z)-9-tetradecen-1-ol acetate (60%), (Z)-9-(E)-12-tetradecadien-1-ol acetate (17%), (Z)-9-(Z)-12-tetradecadien-1-ol acetate (15%), (Z)-9-(E)-11-tetradecadien-1-ol acetate (5%), and (Z)-11-hexadecen-1-ol acetate (3 %) was an effective trap bait for males of this species. The addition of (Z)-9-tetradecen-1-ol to the acetate blends tested resulted in the capture of beet armyworm,S. exigua (Hubner), males which provides further evidence that the alcohol is a pheromone component of this species.     

Feeding and oviposition deterrent activities of flower buds of globemallow,Sphaeralcea emoryi torrey,against boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae)

The globemallow,Sphaeralcea emoryi Torrey, a plant native to Arizona was evaluated as a source of feeding or oviposition deterrents to the boll weevil,Anthonomus grandis Boheman. Feeding and oviposition responses of reproductive weevils to the flower buds and artificial diets spiked with dry powder or extracts of the globemallow buds were determined. Boll weevils were deterred from feeding and ovipositing in the flower buds unless the calyxes were removed. Male and virgin female weevils were discouraged from feeding as much as gravid weevils. Secondary chemicals in the flower buds served primarily as feeding deterrents but also prevented oviposition. The concentration of these chemicals was highest in the calyxes of the buds, and potent deterrent activity could be extracted from the calyxes with methanol. Boll weevils were able to perceive the deterrents by contact chemosensory organs on the antennae, maxillary palps and labial palps.