Search

Statistical study for the weight of a bovine carcass as function of the thickness of the thight, length of a carcass and sex. Four hundred cattle coming from four different farms and dealt in proportionnal number for each sex, are used to study the influence of the height, the thickness of the thigh and sex on the weight for to appreciate a carcass. Animals were killed in Lubumbashi (Zaire) by the S.A.B. society. The weight is the most important variable for the cow's choice in the commercial channel. In this study, other criteria of appreciation are used for giving a better decision for the butcher.

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica . This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow ( Salix sp., Salicaceae), to birch ( Betula rotundifolia , Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica : 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA-seq. Differential expression analyses in the antennae revealed significant upregulation of one minus-C OBP ( Clap OBP27) and one CSP ( Clap CSP12) in the willow feeders. In contrast, one OR ( Clap OR17), four minus-C OBPs ( Clap OBP02, 07, 13, 20), and one plus-C OBP ( Clap OBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow-specific), both plant species differed mainly in their emission rate of terpenoids such as ( E , E )-α-farnesene (high in willow) or 4,8-dimethylnona-1,3,7-triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an "olfactory bridge" for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus-C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.

Charidotella ambita offers a unique opportunity for unambiguously locating its gold reflector by comparing the structure of reflecting and non-reflecting cuticle of the elytron and pronotum. Using light microscopy and TEM, the reflector was located underneath the macrofiber endocuticle just above the epidermis. The reflector is a multilayer comprising up to 50 bilayers alternating high and low density layers parallel to the surface of the cuticle. It is chirped, i.e., showing a progressive decrease in layer thickness from approximately 150 nm-100 nm across its depth. The high density layers in contact with the endocuticle fuse to the last macrofiber when the reflector is interrupted by a trabecula, demonstrating their cuticular nature. Simulated reflectance spectra from models of the multilayer matched the reflection spectra measured on the major gold patch of the elytron of living specimens. Previous reports in adult insects exhibiting metallic colors located their reflector in the upper strata and structures of the cuticle, i.e., epicuticle, exocuticle, scales and hairs. Thus, the endocuticular location of the reflector in C. ambita (and other tortoise beetles) appears unique for adult insects. Gold reflection appears in C. ambita only when the synthesis of the macrolayer endocuticle is complete, which may take up to 2 weeks. The development of the gold reflector coincides with the start of mating behavior, possibly suggesting a signaling function in conspecific recognition once sexual maturity has been reached., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A summary of literature, documented observations and field studies finds evidence that mothers actively defend offspring in at least eight species and three genera of Neotropical Chrysomelinae associated with two host plant families. Reports on three Doryphora species reveal that all are oviparous and feed on vines in the Apocyanaceae. Mothers in the two subsocial species defend eggs and larvae by straddling, blocking access at the petiole and greeting potential predators with leaf-shaking and jerky advances. A less aggressive form of maternal care is found in two Platyphora and four Proseicela species associated with Solanaceae, shrubs and small trees. For these and other morphologically similar taxa associated with Solanaceae, genetic distances support morphology-based taxonomy at the species level, reveal one new species, but raise questions regarding boundaries separating genera. We urge continued study of these magnificent insects, their enemies and their defenses, both behavioral and chemical, especially in forests along the eastern versant of the Central and South American cordillera.

Larvae of the Chrysomelina species Phaedon cochleariae, Hydrothassa marginella, Phratora vulgatissima, Gastrophysa viridula, Gastrophysa atrocyanea, Gastrophysa cyanea and Gastrophysa polygoni produce the iridoid chrysomelidial (1) to defend themselves against predators. Feeding experiments with a deuterated precursor ([(2)H(5)]8-hydroxygeraniol 9) and in vitro isotope exchange experiments with defensive secretion in (2)H(2)O revealed differences in the cyclisation of the ultimate precursor 8-oxogeranial (8) to 1, between members of the genus Gastrophysa and all other species. In P. cochleariae, H. marginella and P. vulgatissima 1 is most likely produced by a Rauhut-Currier-type cyclisation via a "transoid dienamine", with loss of a single deuterium atom from C(4) of the precursor. In contrast, members of the genus Gastrophysa cyclise 8 via a "cisoid dienamine" intermediate, with exchange of all three deuterium atoms from the methyl group at C(3). To study whether the different cyclisation modes influence the stereochemistry of 1, the absolute configuration of 1 of the larvae was determined by GC-MS on a chiral column. In accordance with literature (J. Meinwald, T. H. Jones, J. Am. Chem. Soc. 1978, 100, 1883 and N. Shimizu, R. Yakumaru, T. Sakata, S. Shimano, Y. Kuwahara, J. Chem. Ecol. 2012, 38, 29), we found (5S,8S)-chrysomelidial (1) in H. marginella and P. vulgatissima, but P. cochleariae and all investigated members of the genus Gastrophysa synthesise (5R,8R)-chrysomelidial (1)., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We conducted a phylogeographic study on the cold-adapted leaf beetle Chrysomela lapponica, that feeds on willow or birch, by sampling several populations throughout most of the geographic distribution of the species, and by sequencing for each individual one mitochondrial and two nuclear DNA fragments. Patterns of DNA sequence variation from the mitochondrial and nuclear loci, as displayed in the median-joining networks, appear to display contradicting historical signal: a deep genealogical divergence is observed with the mitochondrial genome between the Alpine population and all other populations found in the Euro-Siberian distribution of the species, that is completely absent with both nuclear loci. We use coalescence simulations of DNA sequence evolution to test the hypothesis that this apparent conflict is compatible with a neutral model of sequence evolution (i.e., to check whether the stochastic nature of the coalescence process can explain these patterns). Because the simulations show that this is highly unlikely, we consider two alternative hypotheses: (1) introgression of the mitochondrial genome of another species and (2) the effect of natural selection. Although introgression is the most plausible explanation, we fail to identify the source species of the introgressed mitochondrial genome among all known species closely related to C. lapponica. We therefore suggest that the putative introgression event is ancient and the source species is either extinct or currently outside the geographic range of C. lapponica explored in this study. The observed DNA sequence variation also suggests that a host-plant shift from willow to birch has occurred recently and independently in each of the three birch-feeding populations. This emphasizes further the relative ease with which these beetles can escape their ancestral host-plant specialization on willow, but shows at the same time that host-plant shifts are highly constrained, as they only occur between willow and birch., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glandular chemical defence relying on the action of salicylaldehyde is characteristic for Chrysomela leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in Chrysomela species. We here identified and characterized SAO of Phratora vitellinae, the only species outside the genus Chrysomela that produce salicylaldehyde as a defensive compound. Although Chrysomela and Phratora are not closest relatives, their SAOs share glucose-methanol-choline oxidoreductase (GMC) affiliation, a specific GMCi subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both Chrysomela and Phratora. Closely related species of Chrysomela and P. vitellinae use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.

Chrysomelid leaf beetles use chemical defenses to overcome predatory attack and microbial infestation. Larvae of Chrysomela lapponica that feed on willow sequester plant-derived salicin and other leaf alcohol glucosides, which are modified in their defensive glands to bioactive compounds. Salicin is converted into salicylaldehyde by a consecutive action of a β-glucosidase and salicyl alcohol oxidase (SAO). The other leaf alcohol glucosides are not oxidized, but are deglucosylated and esterified with isobutyric- and 2-methylbutyric acid. Like some other closely related Chrysomela species, certain populations of C. lapponica shift host plants from willow to salicin-free birch. The only striking difference between willow feeders and birch feeders in terms of chemical defense is the lack of salicylaldehyde formation. To clarify the impact of host plant shifts on SAO activity, we identified and compared this enzyme by cloning, expression, and functional testing in a willow-feeding and birch-feeding population of C. lapponica. Although the birch feeders still demonstrated defensive gland-specific expression, their SAO mRNA levels were 1,000-fold lower, and the SAO enzyme was nonfunctional. Obviously, the loss of catalytic function of the SAO of birch-adapted larvae is fixed at the transcriptional, translational, and enzyme levels, thus avoiding costly expression of a highly abundant protein that is not required in the birch feeders.

Larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) sequester characteristic O-glucosides from the leaves of their food plants, namely Betula and/or Salix The present study focuses on birch-feeding larvae of C. lapponica from the Altai region in East Kazakhstan. As in other sequestering leaf beetle larvae, the compounds are transported intact via different membrane barriers into the defensive system, followed by glucoside cleavage and subsequent transformations of the plant-derived aglycones. Unlike previous studies with model compounds, we studied the sequestration of phytogenic precursors by analyzing the complex pattern of glucosides present in food plant Betula rotundifolia (39 compounds) and compared this composition with the aglycones present as butyrate esters in the defensive secretion. In addition to the analytic approach, the insect's ability, to transport individual glucosides was tested by using hydrolysis-resistant thioglucoside analogs, applied onto the leaf surface. The test compounds reach the defensive system intact and without intermediate transformation. No significant difference of the transport capacity and selectivity was observed between larvae of birch-feeding population from Kazakhstan, and previous results for larvae of birch-feeding population from the Czech Republic or willow-feeding populations. Overall, the transport of the phytogenic glucosides is highly selective and highly efficient, since only minor compounds of the spectrum of phytogenic glucoside precursors contribute to the limited number of aglycones utilized in the defensive secretion. Interestingly, salicortin 44 and tremulacin 60 were found in the leaves, but no aldehyde or esters of salicylalcohol. Surprisingly, we observed large amounts of free glucose, together with small amounts of 6-O-butyrate esters of glucose (27a/b and 28a/b).

Few studies to date have investigated the impact of Pleistocene climatic oscillations on the genetic diversity of cold-adapted species. We focus on the geographic distribution of genetic diversity in a Euro-Siberian boreo-montane leaf beetle, Gonioctena pallida. We present the molecular variation from three independent gene fragments over the entire geographic range of this insect. The observed sequence variation identifies a genetic diversity hot spot in the Carpathian Mountains, in central Europe, which reveals the presence of (1) an ancestral refuge population or (2) a secondary contact zone in this area. Modeling of population evolution in a coalescent framework allowed us to favor the ancestral refuge hypothesis. These analyses suggest that the Carpathian Mountains served as a refuge for G. pallida, whereas the rest of the species distribution, that spans a large portion of Europe and Asia, experienced a dramatic reduction in genetic variation probably associated to bottlenecks and/or founder events. We estimated the time of isolation of the ancestral refuge population, using an approximate Bayesian method, to be larger than 90,000 years. If true, the current pattern of genetic variation in this cold-adapted organism was shaped by a climatic event predating by far the end of the last ice age.

The larval defenses of chrysomeline leaf beetles comprise components that are either synthesized de novo or sequestered from their food plants. Both biosynthetic modes are based on glucosides that serve as substrates and forms of transport. The defensive glands import the compounds through highly selective glucoside transporters from a circulating pool in the hemolymph. Here we address the selectivity of the different transport systems with larvae of Chrysomela populi, an obligate sequestering species, and with larvae of Phaedon cochleariae, producing monoterpene [corrected] iridoids. Both species possess an interconnected network of transport systems for uptake and excretion. The glucosides are imported by the gut membrane with low selectivity. Their excretion by the Malpighian tubules is similarly unselective, but the uptake of the glucosides from the hemolymph into the defensive system is specific. Only the genuine glucoside precursors made de novo or sequestered from the plant are imported. The successful combination of the precursor-adapted pathways of excretion and defense has probably allowed many leaf beetle species to adaptively radiate onto, and coevolve with plants that offer appropriate glucoside precursors.

The tortoise beetle Charidotella egregia is able to modify the structural color of its cuticle reversibly, when disturbed by stressful external events. After field observations, measurements of the optical properties in the two main stable color states and scanning electron microscope and transmission electron microscope investigations, a physical mechanism is proposed to explain the color switching of this insect. It is shown that the gold coloration displayed by animals at rest arises from a chirped multilayer reflector maintained in a perfect coherent state by the presence of humidity in the porous patches within each layer, while the red color displayed by disturbed animals results from the destruction of this reflector by the expulsion of the liquid from the porous patches, turning the multilayer into a translucent slab that leaves an unobstructed view of the deeper-lying, pigmented red substrate. This mechanism not only explains the change of hue but also the change of scattering mode from specular to diffuse. Quantitative modeling is developed in support of this analysis.

The adults of the leaf beetle Platyphora kollari (Chrysomelidae) are able to metabolise the oleanane triterpene beta-amyrin (1) into the glycoside 3-O-beta-D: -glucopyranosyl-(1-->4)-beta-D: -glucuronopyranosyl-hederagenin (2) that is stored in their defensive glands. The aim of this study was to test the hypothesis that oleanolic acid (3) is an intermediate in the conversion of 1 into 2 and to check whether the sequestration of pentacyclic triterpenes is selective in favour of beta-amyrin (1). To this end, adults of P. kollari were fed with Ipomoea batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]oleanolic acid or [2,2,3-(2)H(3)]alpha-amyrin and the secretion of their defensive glands analysed by HPLC-ESIMS. The data presented in this work indicated that the first step of the transformation of beta-amyrin (1) into the sequestered glycoside 2 is its oxidation into oleanolic acid (3) and that this conversion is selective but not specific in favour of beta-amyrin (1).

Methanolic extracts of soldiers of Nasutitermes guayanae and N. surinamensis have been shown to contain complex mixtures of diterpenes and monoterpenes. Eighteen diterpenes have been isolated and identified; twelve of them are previously known nasute termite diterpenes, while six are new trinervitene diterpenes. 2alpha,9beta-Dihydroxy-3beta,8beta-oxido-1(15)-trinervitene has been isolated from N. guayanae, while 3alpha,14alpha-diacetoxy-2beta-hydroxy-1(15),8(19),9-trinervitatriene, 14alpha-acetoxy-2beta,3alpha-dihydroxy-1(15),8(19),9-trinervitatriene, 2beta,3alpha-diacetoxy-11beta,14alpha-dihydroxy-1(15),8(19)-trinervitadiene, 9alpha,14alpha-diacetoxy-2beta,3alpha-dihydroxy-1(15),8(19)-trinervitadiene, and 2beta,9alpha,14alpha-triacetoxy-3alpha-hydroxy-1(15),8(19)-trinervitadiene have been isolated from N. surinamensis. Their structures were determined on the basis of their spectroscopic properties.

Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

Several species of Doryphorina leaf beetles from Central- and South America produce oleanane triterpene glycosides in their defensive glands. The presence of pentacyclic triterpenes in insects is intriguing since they lack the key enzymes necessary to synthesize these compounds. Since beta-amyrin is a common constituent of leaf waxes, we hypothesized that these leaf beetles use this compound as a precursor to their oleanane glycosides. To test this hypothesis we first confirmed the presence of beta-amyrin in Ipomoea batatas, the food plant of Platyphora kollari. Next, adults of P. kollari were fed for 10 days with I. batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]beta-amyrin ([2,2,3-(2)H(3)]-1). The secretion from their defensive glands was collected and analyzed by HPLC-ESIMS. The results demonstrated that the secretion of beetles fed with an amount of [2,2,3-(2)H(3)]beta-amyrin corresponding to the quantity of unlabeled (natural) beta-amyrin present in the leaf disks contained on average 5.1% of [2,2,3-(2)H(3)]-3- O-beta- d-glucopyranosyl-(1-->4)-beta- d-glucuronopyranosyl-hederagenin ([2,2,3-(2)H(3)]-2), whereas the secretions of beetles fed with 10 times this amount of [2,2,3-(2)H(3)]beta-amyrin contained on average 23.9% of [2,2,3-(2)H(3)]-2. In both series of experiments, the percentage of labeled versus unlabeled triterpene glycoside in the secretion was positively correlated with the amount of deuterated beta-amyrin ingested. These results demonstrate for the first time that some leaf beetles are able to metabolize a widespread triterpenic constituent of leaf wax into more complex glycosides that are stored in their defensive glands.

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.

Several neotropical leaf-beetles of the genus Platyphora ingest and specifically metabolize plant acquired pyrrolizidine alkaloids (PAs) of the lycopsamine type (e.g., rinderine or intermedine) and enrich the processed alkaloids in their exocrine defensive secretions. In contrast to the related palaearctic leaf beetles of the genus Oreina, which absorb and store only the non-toxic alkaloid N-oxides, Platyphora sequesters PAs exclusively as protoxic tertiary amines. In this study, the ability of P. boucardi larvae to accumulate PAs was investigated. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi larvae, like adult beetles, utilize the two alkaloidal forms with the same efficiency, but accumulate the alkaloid as a tertiary amine exclusively. Ingested rinderine is rapidly epimerized to intermedine, which is localized in the hemolymph and all other tissues; it is also detected on the larval surface. Like adults, larvae are able to synthesize their own alkaloid esters (beetle PAs) from orally administered [14C]retronecine and endogenous aliphatic 2-hydroxy acids. These retronecine esters show the same tissue distribution as intermedine. A long-term feeding experiment lasting for almost four months revealed that retronecine esters synthesized from [14C]retronecine in the larvae are transferred from larvae via pupae into the exocrine glands of adult beetles. Pupae contain ca. 45% of the labeled retronecine originally ingested, metabolized, and stored by larvae; ca. 12% of larval radioactivity could be recovered from the defensive secretions of adults sampled successively over two and a half months. Almost all of this radioactivity is found in the insect-made retronecine esters that are highly enriched in the defensive secretions, i.e., more than 200-fold higher concentration compared to pupae.

Larvae of a number of chrysomelid leaf beetles sequester phenol glucosides such as salicin from their food plants, i.e. Salix and Populus spp. Salicin is hydrolyzed in the glandular reservoir of the defensive glands. The resulting salicyl alcohol (saligenin) is oxidized by an extracellular oxidase. The product salicylaldehyde accumulates as major defensive compound. The secretions from Chrysomela populi and Phratora vitellinae were preserved in saturated ammonium sulfate solution and subjected to micro-purification of the oxidase by means of electrophoretic methods. The enzyme from P. vitellinae has a native M(r) of 334,000 and a subunit M(r) of 79,000 indicating a tetrameric enzyme. The isoelectric points of the enzymes from C. populi and P. vitellinae are at pH 5.4 and 5.2, respectively. In the oxidation of salicyl alcohol oxygen functions as electron acceptor yielding hydrogen peroxide as product. Hydrogen peroxide does not accumulate in native secretions but appears to be degraded most likely by a catalase. The oxidases from the two species show broad pH optima in the range 5.5 to 6.5, they oxidize salicyl alcohol as main substrate. Minor substrates are several ortho-substituted and to a lesser extent meta- but not para-substituted benzyl alcohols. In the presence of 8-hydroxygeraniol only trace amounts of the respective aldehyde are formed. The Km values of salicyl alcohol are 132 mM (C. populi) and 63 mM (P. vitellinae). The extracellular enzyme, which is functionally related to fungal aryl alcohol oxidase (EC 1.1.3.7) and vanillyl alcohol oxidase (EC 1.1.3.38) was named salicyl alcohol oxidase. The continuous formation of salicylaldehyde in the glandular reservoir can be compared to the operation of an enzyme reactor. Due to its low aqueous solubility the produced aldehyde steadily leaves the aqueous reaction fluid and builds up an organic phase which may account for 15% of the total liquid volume of the secretion.

In vitro experiments using [1-(14)C] and [2-(14)C]acetate were devised to study the biosynthesis of the defensive coccinellid alkaloids adaline and coccinelline in Adalia 2-punctata and Coccinella 7-punctata, respectively. The labelled alkaloids obtained in these experiments had a specific activity about ten times higher than that of the samples obtained in feeding experiments. This in vitro assay has enabled us to demonstrate that these two alkaloids are most likely biosynthesised through a fatty acid rather than a polyketide pathway, that glutamine is the preferred source of the nitrogen atom and that alkaloid biosynthesis takes place in the insect fat body.

Platyphora leaf beetles form a vast group of tropical species each feeding on a restricted set of host plants and exhibiting bright coloration warning predators against their chemical protection. These beetles offer an exceptional opportunity for understanding the evolution of phytochemical sequestration. Indeed, qualitative studies of defensive secretions indicate that Platyphora species acquire toxicity via sequestration of plant secondary metabolites. All produce pentacyclic triterpene saponins from sequestered plant amyrins, but our analyses also indicate that many Platyphora species produce a dual chemical defence, that is, they are additionally protected by lycopsamine-type pyrolyzidine alkaloids that they also sequester from their host. This paper reports on the evolution of chemical defence and host affiliation in Platyphora leaf beetles as reconstructed on a molecular phylogeny of mitochondrial and nuclear DNA sequences. The analyses indicate that dual sequestration could be the key mechanistic means by which transitions among ecological specializations (i.e. restricted host-plant affiliations) are made possible.

Platyphora boucardi leaf-beetles sequester tertiary pyrrolizidine alkaloids of the lycopsamine type acquired from their host-plant Prestonia portobellensis (Apocynaceae) and synthesize their own alkaloids from exogenous retronecine and aliphatic 2-hydroxy acids. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi sequesters both alkaloidal forms with the same efficiency, but accumulates exclusively tertiary alkaloids. There is no substantial alkaloid accumulation in the body outside the defensive glands. Feeding studies with [2H][14C]rinderine confirmed that P. boucardi specifically epimerizes rinderine to its stereoisomers intermedine and lycopsamine. Feeding studies with [2H][14C]retronecine proved the ability of P. boucardi to synthesize O7- and O9-(2-hydroxyisovaleryl)-retronecine and O7-lactyl-O9-(2-hydroxyisovaleryl)-retronecine. Both, alkaloids of the lycopsamine type and self-synthesized retronecine esters accumulate in the defensive secretions at concentrations up to 38 mM and 33 mM, respectively. The different biochemical strategies to maintain pro-toxic pyrrolizidine alkaloids and to prevent self-poisoning, developed by specialized insects, are compared. There are two major findings: (1) the chemical defense mediated by plant acquired pyrrolizidine alkaloids in the taxonomically related palaearctic Oreina and neotropical Platyphora leaf beetles have been evolved independently, since the biochemical mechanisms of storing and maintaining the alkaloids is completely different in the two genera; (2) unexpected parallels exist between taxonomically unrelated Coleoptera and Lepidoptera in their ability to synthesize the same retronecine esters and to catalyze the same site-specific epimerizations of the lycopsamine stereoisomers.

A method has been developed to assign the absolute configuration and enantiomeric excess of anabasine based on small amounts of material (in the microgram range), by derivatization with (+)-menthylchloroformate followed by capillary GC analysis of the resulting carbamate(s). This method was applied to three samples of anabasine isolated from Messor and Aphaenogaster ants. In Messor sanctus, only (2'S)-anabasine was present, whereas in Aphaenogaster subterranea and A. miamiana (2'S)-anabasine was determined to have an ee of 78 and 24%, respectively.

Combination of molecular phylogenetic analyses of Chrysomelina beetles and chemical data of their defensive secretions indicate that two lineages independently developed, from an ancestral autogenous metabolism, an energetically efficient strategy that made the insect tightly dependent on the chemistry of the host plant. However, a lineage (the interrupta group) escaped this subordination through the development of a yet more derived mixed metabolism potentially compatible with a large number of new host-plant associations. Hence, these analyses on leaf beetles document a mechanism that can explain why high levels of specialization do not necessarily lead to "evolutionary dead ends."

Secretions of the pronotal and elytral glands of adults of the chrysomelid beetle Platyphora opima from Panama have been shown to contain two oleanane triterpene saponins: the known 3-O-beta-D-glucopyranosyl-(1-->3)-beta-D-glucuronopyranosyl-oleano lic acid-28-O-beta-D-glucopyranoside and compound 1, whose structure was established as 3-O-beta-D-glucopyranosyl-(1-->3)-beta-D-glucuronopyranosyl-29- hydrox yoleanolic acid-28-O-beta-D-glucopyranoside by a combination of 1D and 2D NMR methods (COSY, HMQC, HMBC, and TOCSY) and FABMS. The secretions also contained N,N,N-trimethylcadaverine and its 1, 2-dehydro derivative 3, as well as the nicotinamide derivative 4. Secretions of Desmogramma subtropica, also from Panama, contained as sole triterpene derivative 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-24- hydrox yoleanolic acid (2), together with glutamic acid, glutamine, pyroglutamic acid, and arginine. A mixture of phosphatidylcholines was also present in the secretions of both species.

The secretion of the defensive glands of adults of the chrysomelid beetle Platyphora ligata from Panama has been shown to contain, besides chlorogenic acid (1) and a mixture of phosphatidylcholines, two new oleanane triterpene saponins, named ligatosides A and B. Their structures were established as 3-O-beta-D-glucuronopyranosyl-16alpha,23-dihydroxyoleanol ic acid-28-O-2-(3,4-dimethoxybenzoyl)-beta-D-glucopyranoside (2) and 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucuronopyranosyl-16a lpha, 23-dihydroxyoleanolic acid-28-O-2-(3, 4-dimethoxybenzoyl)-beta-D-glucopyranoside (3), respectively, by a combination of extensive 1D and 2D NMR methods (COSY, HMQC, HMBC, and TOCSY) and FABMS. This is the first report of triterpene saponins in the defensive secretion of an insect.

The optimal foraging theory predicts that colonies of social insects must be able to adjust the intensity of their foraging behaviour as a function of the quality of the food discovered. Here, the mechanisms allowing the regulation of recruitment as a function of food concentration in the ant Myrmica sabuleti were analyzed. Although the total number of foragers engaged in food collection during recruitments increased with increasing concentration of sucrose solutions (0.1 vs. 1 M), neither the proportion of recruiting scouts nor the invitation behaviour performed by the scouts in the nest can explain this relationship. Foragers trail more when coming back from a 1 M than from a 0.1 M sucrose solution. However, this alone cannot explain the collective patterns observed since the mean numbers of workers leaving the nest after the entry of a scout coming back from either 0.1 or 1 M sources were not significantly different. We suggest that a spatial distribution of the foragers in the nest as a function of their motivational state could be part of the regulation process. The ants located near the nest entrance would respond to both low and high trail pheromone signals, but those located deeper in the nest would respond only to high level signals, resulting in higher recruitment rate towards richer sources.

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.

Larval haemolymph of Neobellieria bullata (Insecta, Diptera) is highly toxic to adults of the same species: injection causes instant paralysis to death. Referring to their dramatic effect in adult insects the responsible compounds were designated paralysins. Two paralysins, soluble in organic solvents and heat stable, were chromatographically purified to homogeneity. They were identified by use of mass spectrometry and nuclear magnetic resonance respectively as beta-alanine-tyrosine (beta-Ala-Tyr) and as 3-hydroxy-kynurenine (3-HK). The quantities of beta-Ala-Tyr and 3-HK in the insect appear to increase steadily during larval development, with peak values prior to the pupal stage. These findings may contribute to a better understanding of some aspects of the process of insect metamorphosis. Orienting experiments in mammals suggest that both compounds, when injected intraspinally, are also neurotoxic to rats. In addition, cytotoxicity tests revealed that 3-HK, but not beta-Ala-Tyr is toxic to human neuroblastoma cells, rat primary cortex neurons as well as to rat glial cells.

Leaf beetles in the genus Phratora differ in host plant use and in the chemical composition of their larval defensive secretion. Most species specialize on either poplars or willows (family Salicaceae), but two species feed on birch (family Betulaceae). Phratora vitellinae utilizes salicylates from the host plant to produce its larval secretion, which contains salicylaldehyde, while other Phratora species produce an autogenous secretion. To reconstruct the evolutionary history of host plant use and the larval secretion chemistry in this genus, we sequenced 1383 base pairs of the mt cytochrome oxidase I gene for six European and one North American Phratora species and three outgroup taxa. Bootstrap values of the complete nucleotide sequence were 99-100% for six of eight nodes in the maximum parsimony tree. They were 71% and 77% for the two other nodes. The maximum parsimony tree and the maximum likelihood tree based on nucleotide sequence showed the same relationships as a maximum parsimony tree based on the amino acid sequence. Beetle phylogeny overlapped broadly with host plant taxonomy and chemistry, and it revealed historical constraints influencing host plant use. However, there was one host shift from the willow family (Salicaceae) to the birch family (Betulaceae). The use of host plant phenol glycosides for the larval defensive secretion evolved along the lineage that led to P. vitellinae. Phratora vitellinae feeds on the taxonomically widest range of host plants, which are characterized by moderate to high levels of salicylates. The results support the hypothesis that the use of salicylates for the larval secretion evolved twice independently in chrysomeline leaf beetles., (© 1998 The Society for the Study of Evolution.)

A phylogenetic analysis of the genus Gonioctena (Coleoptera, Chrysomelidae) based on allozyme data (17 loci) and mitochondrial DNA sequence data (three gene fragments, 1,391 sites) was performed to study the evolutionary history of host-plant shifts among these leaf beetles. This chrysomelid genus is characteristically associated with a high number of different plant families. The diverse molecular data gathered in this study are to a large extent congruent, and the analyses provide a well-supported phylogenetic hypothesis to address questions about the evolution of host-plant shifts in the genus Gonioctena. The most-parsimonious reconstruction of the ancestral host-plant associations, based on the estimated phylogeny, suggests that the Fabaceae was the ancestral host-plant family of the genus. Although most of the host-plant shifts (between different host species) in Gonioctena have occurred within the same plant family or within the same plant genus, at least eight shifts have occurred between hosts belonging to distantly related and chemically dissimilar plant families. In these cases, host shifts may have been simply directed toward plant species available in the environment. Yet, given that two Gonioctena lineages have independently colonized the same three new plant families (Salicaceae, Betulaceae, Rosaceae), including four of the same new genera (Salix, Alnus, Prunus, Sorbus), some constraints are likely to have limited the different possibilities of interfamilial host-plant shifts.

Field investigations showed that the ant Lasius fuliginosus combines stable foraging behaviour based on long-term memory and spatial specialization with more flexible actions, based on food recruitment. The use of food recruitment varies according to the season and the type of food. Baits of meat or large prey elicited recruitment during the major part of the year, except in the autumn, while baits of sugar elicited recruitment only in early spring, before the aphid colonies developed. A permanent trunk trail was established after a recruitment towards a newly discovered and stable source of carbohydrate food. This happened in early spring when the food sources were scarce or when spring migrating aphids were produced.

Insect-plant interactions have played a prominent role in investigating phylogenetic constraints in the evolution of ecological traits. The patterns of host association among specialized insects have often been described as highly conservative, yet not all specialized herbivorous insect lineages display the same degree of fidelity to their host plants. In this paper, we present an estimate of the evolutionary history of the leaf beetle genus Oreina. This genus displays an amazing flexibility in several aspects of its ecology and life history: (1) host plant switches in Oreina occurred between plant families or distantly related tribes within families and thereby to more distantly related plants than in several model systems that have contributed to the idea of parallel cladogenesis; (2) all species of the genus are chemically defended, but within the genus a transition between autogenous production of defensive toxins and sequestration of secondary plant compounds has occurred; and (3) reproductive strategies in the genus range from oviparity to viviparity including all intermediates that could allow the gradual evolution of viviparity. Cladistic analysis of 18 allozyme loci found two most parsimonious trees that differ only in the branching of one species. According to this phylogeny estimate, Oreina species were originally associated with Asteraceae, with an inclusion of Apiaceae in the diet of one oligophagous species and an independent switch to Apiaceae in a derived clade. The original mode of defense appears to be the autogenous production of cardenolides as previously postulated; the additional sequestration of pyrrolizidine alkaloids could have either originated at the base of the genus or have arisen three times independently in all species that switched to plants containing these compounds. Viviparity apparently evolved twice in the genus, once without matrotrophy, through a retention of the eggs inside the female's oviducts, and once in combination with matrotrophy. We hypothesize that the combination of autogenous defense and a life history that involves mobile externally feeding larvae allowed these beetles to switch host plants more readily than has been reported for highly conservative systems., (© 1996 The Society for the Study of Evolution.)

The pronotal and elytral defensive secretions of 10Oreina species were analyzed. Species feeding on Apiaceae, i.e.,O. frigida andO. viridis, or on Cardueae (Asteraceae), i.e.,O. bidentata, O. coerulea, andO. virgulata, produce species-specific complex mixtures of autogenous cardenolides.O. melanocephala, which feeds onDoronicum clusii (Senecioneae, Asteraceae), devoid of pyrrolizidine alkaloids (PAs) in its leaves, secretes, at best, traces of cardenolides. Sequestration of host-plant PAs was observed in all the other species when feeding on Senecioneae containing these alkaloids in their leaves.O. cacaliae is the only species that secretes host-derived PA N-oxides and no autogenous cardenolides. Differences were observed in the secretions of specimens collected in various localities, because of local differences in the vegetation. The other species, such asO. elongata, O. intricata, andO. speciosissima, have a mixed defensive strategy and are able both to synthesize de novo cardenolides and to sequester plant PA N-oxides. This allows a great flexibility in defense, especially inO. elongata andO. speciosissima, which feed on both PA and non-PA plants. Populations of these species were found exclusively producing cardenolides, or exclusively sequestering PA N-oxides, or still doing both, depending on the local availability of food-plants. Differences were observed between species in their ability to sequester different plant PA N-oxides and to transform them. Therefore sympatric species demonstrate differences in the composition of their host-derived secretions, also resulting from differences in host-plant preference. Finally, within-population individual differences were observed because of local plant heterogeneity in PAs. To some extent these intrapopulation variations in chemical defense are tempered by mixing diet and by the long-term storage of PA N-oxides in the insect body that are used to refill the defensive glands.

8-Hydroxygeraniol and its 8-O-β-D-glucoside have been found as trace components in the defensive secretions ofPlagiodera versicolora andGastrophysa viridula larvae. This discovery supports the hypothesis that the evolution of the utilization of plant precursors by some chrysomelid species was favored by the plesiomorphic occurrence of aβ-glucosidase and an oxidase in the defensive secretion of iridoid-producing species.

In five New GuineaNasutitermes (N. gracilirostris, N. novarumhebridarum, N. torresi, N. triodiae, and an undescribed speciesN. sp.F), we have detected and identified 10 sesquiterpenes. Eight of these compounds (β)-elemene,β-caryophyllene,α-humulene,α-muurolene,γ-selinene,β-selinene, germacrene-A, and γ2-cadinene) were identified by GC and GC-MS (EI). Two uncommon sesquiterpenes, (5R(*), 7R(*), 10S(*))-selina-4(14),11-diene and (5R(*), 7R(*), 10S(*))-selina-3,11-diene, were identified by GC, GC-MS (EI, CI), GC-FTIR, and mono- and bidimensional NMR. Whereas in most species sesquiterpenes are present in low or trace amounts, inN. novarumhebridarum the sesquiterpenic fraction of soldier frontal gland secretion is equal to that of the monoterpenes.

(6R*)-[(2S*)-2-hydroxyheneicos-12-enyl]-5,6-dihydro-2H-pyran-2-one (1) degree is the major constituent of the secretion of freshly dissected Dufour's gland of the urticating ant Tetramorium aculeatum. In solution, compound 1 is slowly transformed into (1S*,5R*,7S*)-7-(nonadec-10-enyl)-2,6-dioxabicyclo[3.3.1] nonan-3-one (2) degrees on standing. The structures of compounds 1 and 2 have been established on the basis of their spectral and chemical properties. Compound 1 could be responsible for the urticating properties of the ant.

Oreina cacaliae (Chrysomelidae) sequesters in its elytral and pronotal defensive secretion theN-oxides of pyrrolizidine alkaloids (PAN-oxides) from its food plantAdenostyles alliariae (Asteraceae). [(14)C]SenecionineN-oxide was applied for detailed studies of PAN-oxide sequestration. An average of 11.4% of total radioactivity is taken up by individual beetles which had received [(14)C]senecionineN-oxide with their food leaves 8 days before. An average of 28.9% of the ingested radioactivity could be recovered from the defensive secretions collected twice, i.e., 5 and 8 days after tracer feeding. The tracer transfer into the secretion seems to be a slow but progressive process as indicated by the high percentage of tracer still recovered from the secretion sampled after 8 days. Chromatographic analysis revealed that [(14)C]senecionineN-oxide is the only labeled compound in the defensive secretion. Beetles that fed on tertiary [(14)C]senecionine sequestered only trace amounts of radioactivity (exclusively present as labeled IV-oxide) in their secretions.O. speciosissima, a species also adapted to PA containing food plants, was shown to sequester [(14)C]senecionineN-oxide with the same efficiency asO. cacaliae. O. bifrons, a specialist feeding onChaerophyllum hirsutum (Apiaceae), rejected PA treated leaf samples already at very low PA concentrations (10 nmol/leaf piece). In bothO. cacaliae andO. speciosissima, [(14)C]senecionineN-oxide applied by injection into the hemolymph is rapidly transferred into the glands.O. bifrons, not adapted to pyrrolizidine alkaloid containing plants was unable to sequester [(14)C]-senecionineN- oxide in the secretion but rapidly eliminated the tracer with the frass. Again, only traces of labeled [(14)C]senecionineN-oxide were found in the defensive secretions of the two PA adapted species if labeled senecionine was injected. It is suggested that the beetles are adapted to theN-oxide form of PAs, similarly as their food plants, and that they lack the ability to efficientlyN-oxidize tertiary PAs. No indication forde novo PA synthesis by the beetles was found in tracer feeding experiments with the biogenetic PA precursor putrescine.

The soldier frontal gland secretion ofNasutitermes princeps induces strong short-range caste-specific alarm and attraction in both soldiers and workers. Soldiers are excited and patrol the surroundings of the source. The secretion per se does not induce ejection of additional secretion. Large workers of the second stage or older are massively attracted when tested in homogeneous groups. They focus their activities much more accurately than the soldiers around the source. The workers' reaction is less intense in the presence of soldiers. Large and small workers of stage 1 scarcely react at all to the secretion, whether tested in homogeneous or mixed groups. These results suggest the following complementary roles of soldiers and workers in defense. The first line of defense is provided by soldiers, which immobilize and incapacitate mobile enemies with their sticky secretion. Defense then is completed by older large workers as they eliminate the source of disturbance. The absence of reaction of young workers, small or large, confirms previous reports on age polyethism inNasutitermes observed in other contexts: young workers tend to stay in the nest. Alarm reactions are elicited by a source of (+)-α-pinene, the major monoterpene in the secretion, while its enantiomer, almost absent from the secretion, induces a much weaker reaction.

In the Chrysomelinae, it appears that de novo synthesis of chemicals for defense is the primitive state, and the sequestration of plant chemicals for defense the derived state. The derived state evolved through both the morphological and biochemical preadaptiveness of the homologous defensive glands. In the adults, we discuss one unique case of sequestration in exocrine defensive glands of host-plant pyrrolizidine alkaloids byOreina cacaliae. However, hypericin is not sequestered either in the glands or elsewhere in the body ofChrysolina spp. feeding onHypericum, which contradicts an earlier claim. In the larvae, we examine in more detail how the phenolglucoside salicin can be used as the precursor of the salicylaldehyde present in the defensive secretion ofPhratora vitellinae andChrysomela spp. with minimal changes in the biochemical mechanisms involved in the biosynthesis of iridoid monoterpenes in related species.

Ventral glands are common in nematine larvae (Hymenoptera: Symphyta), but they show various degrees of development and are functional for defense only in some species. In those species, volatile irritants are produced which are effective against ants. Alternative or complementary mechanisms against ants are the pubescence ofTrichiocampus spp., the foam pillars constructed byStauronema compressicornis, various movements of the abdomen, which occur independently of the glandular secretion in several species, immobility of the flat larvae ofNematinus luteus, and burrowing within plant tissues in gallicolous larvae or miners. Glandular development is not clearly related to the appearance of the larvae, either cryptic or aposematic. The secretion, even when it is produced in large amounts by species with well-developed glands, is only moderately efficient against great tits. Bright colors are found in gregarious larvae; these were accepted only with reluctance by great tits and sometimes rejected, even species in which the ventral glands are reduced. We suggest that the various volatile irritants secreted by ventral glands are aimed primarily against insects (e.g., ants) and only secondarily against birds.